[A new technique to facilitate donor preparation for DMEK surgery]. / Eine neue Technik zur Optimierung der Spenderpräparation bei der DMEK.

BACKGROUND: Descemet membrane endothelial keratoplasty (DMEK) is becoming more and more the method of choice to treat corneal endothelial diseases in specialized centers. The reasons that prevent this technique from becoming widespread are the delicate donor tissue preparation. By inverting the curvature of the cornea from convex to concave after mounting onto an artificial anterior chamber, we developed a combined manual delamination and hydrodissection technique, which allows a rapid and endothelium-preserving method of separating donor Descemet membranes from the underlying stroma. MATERIAL AND METHODS: Experiments were perfomed with 60 donor corneas that were not suitable for transplantation. Donor age ranged between 42 and 94 years. Two experimental groups were formed: 1 inverse manual delamination (n = 16) and 2 combined manual delamination and hydrodissection (n = 44). All experiments were undertaken by an experienced surgeon who was, however, not experienced with these techniques. We examined the frequency of Descemet membrane rupture as well as the amount of induced endothelial damage (trypan blue staining with quantitative image analysis). RESULTS: Significant lesions of Descemet's membrane that would have led to a loss of the graft occurred in 25% of the manual delamination cases and in 4.5% using the combined technique. Endothelial damage induced by both techniques was low (6 and 5.2%, respectively). CONCLUSION: For DMEK donor preparation, a combination of manual delamination and hydrodissection was shown to be a safe and endothelium-protective technique to separate Descemet membranes from the underlying stroma. A very rapid learning curve for the combination technique is of specific additional interest for beginners in DMEK surgery.

Retrospective contralateral study comparing Descemet membrane endothelial keratoplasty with Descemet stripping automated endothelial keratoplasty.

PURPOSE: In this retrospective study, the visual outcomes and postoperative complications after Descemet stripping automated endothelial keratoplasty (DSAEK) and Descemet membrane endothelial keratoplasty (DMEK) in the fellow eye were compared. The patient's satisfaction was evaluated. METHODS: A retrospective analysis of 10 patients, who underwent DSAEK in one eye and DMEK surgery in their fellow eye, was performed. Intraoperative and postoperative complications were recorded. Visual and refractive outcomes were evaluated, including higher-order aberrations (HOA) and contrast thresholds. A subjective questionnaire was used to evaluate patient satisfaction. RESULTS: Best-corrected visual acuity (BCVA) was significantly better in DMEK when compared with DSAEK (0.16±0.10 vs 0.45±0.58 logMAR, P=0.043). Contrast threshold was significantly higher after DMEK than after DSAEK (0.49±0.23 vs 0.25±0.18, P=0.043). Post-keratoplasty astigmatism, mean spherical equivalent, and HOA did not differ. Nine out of ten patients preferred the DMEK procedure. Visual outcome (4.80±1.14 vs 4.50±1.58, P=0.257), surgery associated pain and burden (DMEK: 1.30±0.48 vs DSAEK: 1.30±0.48, P=1.0), estimated time for recovery and rehabilitation (27.6±54.0 vs 24.9±54.8 days, P=0.173), and mean patient satisfaction (5.40±0.84 vs 5.00±1.05, P=0.257) were evaluated equally. CONCLUSION: Patient satisfaction reached high, equal values after DMEK and after DSAEK. Nevertheless, patients preferred DMEK, if given a choice. Reasons for the preference may include better uncorrected and BCVA, and especially a better contrast sensitivity.

[Endothelial cell loss after retropupillary iris-claw intraocular lens implantation]. / Endothelzellverlust nach retropupillar fixierter Irisklauen-Linse.

BACKGROUND: The aim of this study was to evaluate the indication, visual and refractive outcome, endothelial cell loss and complication rate after implantation of a posterior iris-claw aphakic intraocular lens (IOL). PATIENTS AND METHODS: This retrospective study comprised 62 eyes of 56 patients without adequate capsular support undergoing posterior iris-claw aphakic IOL implantation (Verisyse™/Artisan®) between 2006 and 2012. Mean follow-up was 34 months (range from 13 to 78 months). RESULTS: The IOLs were inserted during primary lens surgery in 11 phakic eyes (17.8 %), during an IOL exchange procedure for dislocated posterior chamber IOLs in 34 eyes (54.8 %), and as a secondary procedure in 17 aphakic eyes (27.4 %). The final best spectacle-corrected visual acuity (BSCVA) in logMAR (mean 0.24 ± 0.45) improved significantly (p < 0.001) compared to the preoperative BSCVA (mean 0.61 ± 0.65). The mean spherical equivalent improved from preoperative 7,25 ± 5,04 diopters (D) (range - 10.25 to + 16.0 D) to - 0.21 ± 1.01 D (range - 4.0 to 3.0 D) postoperatively. Mean central endothelial cell density was 1844 ± 690 cells/mm(2) preoperatively. After surgery mean endothelial cell density decreased statistically not significant with a loss of 5.5 % to 1743 ± 721 cells/mm(2) (p > 0.05) at last follow-up visit. Complications included cystoid macular oedema in 4 eyes (6.4 %), early postoperative hypotony in 2 eyes (3.2 %), pupil ovalisation in 2 eyes (3.2 %), traumatic iris-claw IOL disenclavation in 2 eyes (3.2 %) and spontaneous IOL disenclavation in one eye (1.6 %). CONCLUSIONS: Retropupillar iris-claw IOL provides good visual and refractive outcomes with a low endothelial cell loss and can be used for a wide range of indications in eyes without adequate capsular support.

[Influence of donor lamella thickness on visual acuity after Descemet's stripping automated endothelial keratoplasty (DSAEK)]. / Einfluss der Spenderlamellendicke auf die Sehschärfe nach "Descemet's stripping automated endothelial keratoplasty" (DSAEK).

BACKGROUND: The aim of this study was to evaluate the effect of donor lamella thickness on postoperative visual acuity after Descemet's stripping automated endothelial keratoplasty (DSAEK). MATERIALS AND METHODS: A retrospective analysis of 65 eyes from 61 patients who underwent DSAEK surgery in cases of Fuchs' corneal dystrophy or bullous keratopathy between 2008 and 2011 was performed. The thickness of donor lamella was measured intraoperatively by ultrasonic pachymetry and postoperatively by anterior segment optical coherence tomography (OCT) and correlated to the visual acuity and number of endothelial cells. RESULTS: The donor lamella thickness measured intraoperatively and postoperatively correlated significantly with each other (r = 0.874, p < 0.001). A significant correlation was found between postoperative corneal lamella thickness measured by anterior segment OCT and visual acuity (r = 0.273, p = 0.028) but not between intraoperative donor lamella thickness measured by ultrasonic pachymetry and visual acuity (r = 0.241, p = 0.103). The postoperative endothelial cell number did not show a correlation with either the intraoperatively or the postoperatively measured donor lamella thickness (r = - 0.059, p = 0.731, r = 0.024, p = 0.869, respectively). CONCLUSIONS: Corneal lamella thickness < 120 µm was found to be correlated with a better visual outcome than in cases of thicker corneas > 120 µm. Despite greater difficulty in corneal transplant technique in cases of thinner lamella no increased damage of corneal endothelium was shown. Therefore, DSAEK with corneal lamella thickness < 120 µm is an interesting therapeutic alternative to DMEK.

[A comparative in vitro analysis of primary and immortalized keratocytes]. / Vergleichende In-vitro-Analyse von primären und immortalisierten Keratozyten.

BACKGROUND: The cultivation of primary keratocytes (HCKp) is difficult and influenced by a multitude of factors. In this study it was examined if immortalized keratocytes (HCKi) can replace HCKp in experiments and be useful in the development of a cornea construct. METHODS: HCKp and HCKi were cultivated and incubated for 72 h with benzalkonium chloride (BAC) or cetrimide at concentrations of 40-0.1 microg/ml or 100-0.01 microg/ml. The vitality and the doubling time (tv) were measured. RESULTS: Treatment with 40 or 4 microg/ml BAC as well as 100 or 10 microg/ml cetrimide led to cell death. The tv was shortened in HCKi especially in cells that were treated with BAC, but only HCKp showed a significant loss of vitality. In cells treated with cetrimide the tv increased significantly in both cell lines and no loss of vitality was detected from 0.1 microg/ml onwards in both cell lines. CONCLUSION: HCKi are more resistant and proliferative than HCKp but they can be used in preliminary experiments as an alternative to primary cells in for example toxicity studies if the detectable differences between the two cell lines, such as the capacity for proliferation and reaction to agents are taken into consideration.

[Significance of cyclosporin A absorption for effective immunomodulatory therapy after high-risk keratoplasty]. / Bedeutung der Cyclosporin-A-Absorption für eine effektive immunmodulatorische Therapie nach Hochrisikokeratoplastik.

AIM: Cyclosporin A (CsA) is applied as an immune modulator in transplantation medicine, including high-risk keratoplasty. We examined the C(0) and C(2)-CsA serum levels after high-risk keratoplasty. The rate of so-called low absorbers was determined, and the importance of low absorption for the survival and rejection freedom of corneal grafts was examined. METHODS: Follow-up of 89 high-risk cornea grafts in the patient group (n=32) ranged from 1 to 72 months postoperatively. The evaluation included data about serum levels of CsA C(0) (24 h after oral CsA application) and C(2) (2 h after oral CsA application) and clinical follow-ups. Using statistical methods, CsA C(0) and C(2) levels and clinical data were examined. Low absorbers did not reach the target C(2) levels by CsA dose adjustment. OUTCOMES: High intraindividual and interindividual variance of CsA C(0) and C(2) values was observed in the examined group of patients. A rate of up to 34.4% (n=11) represented the low absorbers. There was no significance of the low-absorption factor for clear graft survival, rejection freedom, or the rate of side effects observed. An acute endothelial rejection was observed in 23% of grafts and caused 37% of graft failures. In the patients without rheumatic corneal ulcers (n=27), 89% of the corneal grafts remained clear after 12 months, and 52% remained clear after 36 months. CONCLUSIONS: In our study, with a low number of patients and multiple cofactors, the influence of low-absorption CsA on the clear corneal graft survival and rejection rates could not be proved statistically. Due to the specific immune status of the cornea, the influence of low absorption may be lower than in organs such as the kidney and liver. In our study, the C(2) serum level of CsA after high-risk keratoplasty did not provide any helpful information about the prognosis of the corneal grafts or CsA treatment monitoring.

[A new posterior chamber intraocular lens for sutureless iris-fixated ciliary sulcus implantation in aphakic eyes without capsular support]. / Neue Hinterkammerlinse zur sulkusgestützten und nahtlos irisfixierten Implantation in aphake Augen ohne Kapselstrukturen.

PURPOSE: To avoid anterior chamber lens implantation in aphakic eyes without capsular or zonular support, the laborious scleral fixation of a standard posterior chamber intraocular lens (IOL) is widely used, despite a large range of possible complications (macular edema, IOL tilt/decentration, suture erosion, vitreous hemorrhage). A sutureless iris-fixed ciliary sulcus implantation of a posterior chamber IOL (Binder-IOL, Fa. Iolution, Itzehoe) designed especially for such cases represents an alternative. METHOD: At the end of both haptics of this IOL, with a 6 mm optic zone, a T-shaped anchor is mounted at an angle of 45 degrees from the optical plane. These anchors are inserted from behind the iris into peripheral iridotomies performed preoperatively with a YAG-laser or intraoperatively with a vitrectomy cutter. The long, C-shaped haptics are thus secured in the ciliary sulcus. RESULTS: After slight modification of the technique, the implantation can be performed securely and reproducibly. In the 22 implantations performed to date, the IOL was well centered and stable, even in cases with only residual iris stroma. If the iridotomies are been performed too centrally, an ovalisation of the pupil may occur. No further side effects have been observed. CONCLUSION: The technique of sutureless sulcus fixation presented here leads to less complications than scleral suture fixation. A prerequisite for safe implantation of the anchors is good visibility of the peripheral iris. The implantation of the Binder-IOL is especially suitable for aphakic eyes with a loosened iris diaphragm.

[Effect of suramin on proliferation and migration of lens epithelial cells in vitro]. / Effekt von Suramin auf Proliferation und Migration von Linsenepithelzellen in vitro.

BACKGROUND: New approaches for the treatment of secondary cataract focus on a pharmacological therapy to prevent posterior capsule opacification. In this study we investigated the effect of the naphthylurea suramin in vitro in a cell culture model. MATERIAL AND METHODS: Cell proliferation was measured using a proliferation assay and cell migration with a migration assay. Analyses of suramin toxicity were carried out using trypan blue staining. RESULTS: A significant dose-dependent effect of suramin on the inhibition of LEC proliferation and migration was found even after short (1 or 2 h) incubation times ( p<0.01). Cell lysis reflecting a cytotoxic effect of suramin was not found even with high concentrations. However, preincubation of lens capsules with suramin did not prevent LECs from migrating compared to control cultures. CONCLUSION: These results demonstrate the efficiency of suramin in vitro to inhibit migration of HLEC which can be explained by blocking growth factor-mediated effects on the cells. However, further investigations on the pharmacokinetic properties of suramin and possible side-effects on other intraocular tissues must follow.

Intracellular signaling pathway of FGF-2-modulated corneal endothelial cell migration during wound healing in vitro.

After wounding, the corneal endothelium heals primarily by migration of adjacent cells into the denuded wound area. In this study, it has been attempted to identify elements of the intracellular signaling pathway activated through basic Fibroblast Growth Factor (FGF-2)- and Protein Kinase C (PKC)-modulated migration, using specific inhibitors and stimulators of second messengers in a cell culture model. Bovine corneal endothelial cells (BCEC) were grown to confluency and experiments performed with first passage cells under serum-free conditions. A central circular 'wound' was made with a specially designed trephine. In different experiments, cells were incubated with either FGF-2 (10 ng ml(-1)), pertussis toxin (PTX; 1-50 ng ml(-1)), phorbol 12-myristate 13-acetate (PMA; 50 ng ml(-1)), 2,4'-di-bromoacetophenone (DAP; 5 microM), 1-(5-iosquinolinesulphonyl)-2-methyl-piperazine dihydrochloride (H7; 10 microM), indomethacin (5 ng ml(-1)), nordihydroguaiaretic acid (NDGA; 10 ng ml(-1)), 2-(4-morpholinyl)-8-pheny-4H-1-benzopyran-4-one (LY294002; 10 microM) or different combinations of these agents. Unsupplemented cultures served as controls. Migration was quantitated by counting the cells inside the denuded area in one randomly chosen section from the wound edge 72 hr after wounding. Cell toxicity was determined with the trypan blue exclusion test. Results were statistically analysed by Student's t-test. FGF-2 and PMA (a protein kinase C activator) both stimulated migration of endothelial cells at 2.2- and 3.1-fold, respectively. The PLA(2) inhibitor DAP and the PKC inhibitor H7 both significantly reduced PMA-stimulated migration to control levels but had no effect (DAP) or even stimulated (H7) FGF-2-modulated migration. PTX did not affect FGF-2-stimulated migration. The phosphoinositol (3)-kinase inhibitor LY294002 significantly reduced FGF-2-mediated stimulation of endothelial migration similar to the rate of control cultures. LY294002 had no effect when applied together with PMA. The cyclooxygenase inhibitor indomethacin did not influence migration rates of the cells added either alone or in combination with PMA and FGF-2, respectively. The lipoxygenase inhibitor NDGA significantly reduced the number of migrating cells in cultures with no other supplements, or of those supplemented with either PMA or FGF-2. FGF-2-induced endothelial migration in vitro is not dependent on PKC/PLA(2) or pertussis-toxin sensitive G-protein pathways but rather requires activation of a phosphoinositol (3)-kinase-like enzyme and/or arachidonic acid release with subsequent liberation of lipoxygenase products. Independent of FGF-2, PKC is a major intracellular effector of corneal endothelial migration activity after wounding and stimulates migration via the PLA(2)-dependent generation of lipoxygenase metabolites.

The role of endogenous growth factors to support corneal endothelial migration after wounding in vitro.

Previous studies have shown that corneal endothelial cells contain mRNA and protein of various growth factors. However, the role of these endogenous growth factors in corneal endothelial wound healing is not fully elucidated. In the present study, we investigated the role of endogenous factors and several growth factor inhibitors on migration of corneal endothelial cells in an in vitro model of wound healing. Bovine corneal endothelial cells (BCEC) were grown to confluency and experiments performed at passage 2 under serum reduced [2% fetal calf serum (FCS)] conditions. A central circular 'wound' (5 mm diameter) was made with an especially designed trephine. In different experiments, cells were incubated over different time periods (1-72 hr) either with the cellular debris produced by the wounding procedure or with previously prepared endothelial cell lysates (protein content 50-500 microg ml(-1)). Additionally, purified bovine polyclonal anti-BFGF antibodies (Ab) (4.5-27 microg ml(-1)), suramin (0.5 m M) or anti-FGF receptor-Ab (1 microg ml(-1)) were added to both experimental approaches, respectively. Migration was quantitated by counting the cells inside the denuded area in four different sections from the wound edge after 5 days. Cellular migration of cells adjacent to the wound was significantly stimulated by factors released during wounding or by endothelial cell lysates at protein concentrations >100 microg ml(-1). This increase in migrating cells was partially inhibited when the anti-bFGF antibody was incubated with the cell debris or the lysates. The addition of suramin at 0.5 m M almost completely blocked the migration activity. Incubation of the anti-FGF-receptor antibody prior to and >5 hr after wounding significantly reduced migration to nearly 50% of the rate in control cultures (P<0.001). In the present study, we demonstrate that intracellular growth factors released from corneal endothelial cells enhance the migration of surviving cells in vitro. The strong inhibitory effect of suramin indicates a major role of heparin-binding growth factors for cellular migration. bFGF and the regulation of bFGF-receptor expression on cells at the wound margin seem to be of crucial importance for the wound healing process.

Corneal endothelial toxicity of different lidocaine concentrations.

PURPOSE: To examine the potential damaging effect on the corneal endothelium of unpreserved lidocaine in concentrations of 1%, 5%, and 10%. SETTINGS: Department of Ophthalmology, Charité Medical Faculty, Humboldt University, Berlin, Germany. METHODS: Experimental porcine corneas (n = 18) were exposed to 100 microL of unpreserved lidocaine hydrochloride at concentrations of 1%, 5%, and 10% for 60 minutes. Additional corneas (n = 6) were treated with lidocaine hydrochloride 1% for 30 minutes to simulate clinical conditions. Balanced salt solution (BSS((R))) served as a control to evaluate corneal endothelial cell damage using Janus Green photometry. Morphology, damage pattern, and changes in the ultrastructural appearance of corneal endothelial cells were examined by light and scanning electron microscopy. RESULTS: Lidocaine 1% used for 30 or 60 minutes did not cause significantly more corneal endothelial damage (mean 3.00% +/- 0.76% [SD] and 3.26% +/- 1.00%, respectively) than in the control group (mean 3.32% +/- 0. 86%) (P >.01). Significant corneal endothelial cell loss was observed with lidocaine 5% (mean 10.7% +/- 6.4%) (P <.001) and lidocaine 10% (42.3% +/- 17.0%) (P <.001). CONCLUSION: Experimental exposure of corneal endothelial cells to higher concentrations of lidocaine resulted in significant cell loss, indicating that the 1% concentration only should be used clinically.

The intrastromal corneal ring in clinical refractive surgery: reference to results in rabbit eyes.

BACKGROUND: The intrastromal corneal ring (ISR) is a refractive device recently introduced for clinical application that is implanted in the mid-peripheral corneal stroma in order to correct myopia without invasion of the central optical zone. First clinical results of intracorneal ring segment implantation were published recently. These results reveal striking similarities to our own experimental data, only briefly published up to now. The aim of this study is to present the refractive and histopathological data of ISR implants in rabbits and to compare these results with the clinical data actually available. METHODS: Expansion/constriction effects were evaluated with a ring of constant size (7.5 x 0.5 x 0.2 mm) in channels of 7.0, 7.5, or 8.0 mm in diameter, volume effects by implantation of 7.5-mm rings with varying thickness (0.2, 0.3, 0.4 mm) into a channel of 7.5 mm, respectively. Refractive power was measured preoperatively and at day (D) 7, D14, D30 for the first and the second experiment, plus D60, D90 for the second experiment. Histological evaluations of the induced morphological changes were additionally performed at all time intervals. RESULTS: Significant (P<0.05) flattening of the cornea was obtained in all but the first (constant ring, 7.0-mm channel) implants postoperatively at D7 and/or D14, with mean dioptric changes up to -5.03+/-2.92 compared with controls. However, from D30 on, there was no statistically significant difference between operated and control eyes. Biomicroscopy and histology of the implanted eyes revealed good biocompatibility, with only rare major complications such as stromal abscess or massive neovascularization. CONCLUSIONS: Although our implantation technique differs slightly from that employed in the recent FDA studies, our results tend to confirm the maximal achievable refractive change of about -5 dpt with this procedure. Furthermore, this study is the first to demonstrate that in rabbits, ISR implantation has only a shortterm effect on refractive power. Our results indicate that long-term refractive follow-up may be necessary in human eyes prior to the introduction of ISRs as a routine procedure in refractive surgery.