Peter Rieck

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 &mgr;L 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®) 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.

Corneal wound healing modulation using basic fibroblast growth factor after excimer laser photorefractive keratectomy.

Photorefractive keratectomy with the 193-nm excimer laser is one of the most promising innovations in refractive surgery. However, routine clinical application is hindered by two main obstacles, i.e., postoperative subepithelial opacity and possible regression of the refractive result. Because we have previously demonstrated beneficial effects of basic fibroblast growth factor (bFGF) on corneal epithelial healing in different in vivo models, we investigated the influence on both epithelial and stromal healing of topical bFGF after excimer laser keratomileusis in a rabbit model. After 7-mm circular deepithelialization, the eyes of 24 New Zealand White rabbits received identical deep stromal laser ablations (depth 50 µm, 6 D, diameter 5 mm) and were randomly assigned to one of four treatment groups [control (phosphate-buffered saline, PBS), bFGF 10 µg/application, dexamethasone 0,1%, bFGF + dexamethasone, n=12 eyes/group]. All treatments were administered four times daily—bFGF until complete epithelial healing, dexamethasone and PBS until 3 months postsurgery. Wound surface regression on time was determined by means of computer-assisted image analysis of fluorescein-stained corneas. Corneal opacity was observed biomicroscopically and graded using a previously established scoring system. After bFGF application for 2-3 days only, a highly significant acceleration in epithelial wound healing speed was found compared with the rate of the other three treatment groups (p<0.001). A combined therapy (bFGF + steroid) had no effect on the healing rate. Compared with control values, mean scores for subepithelial haze in the other groups were found to be nearly 50% lower during the first 2 postoperative months. No significant difference could be seen between eyes that received bFGF, dexamethasone, or bFGF + steroid. These results demonstrate for the first time the efficiency of a growth factor in modulating the wound healing response after excimer laser photoablation. Also with regard to possible adverse reactions, a short-term and low-dose bFGF application seems advantageous over a continuous treatment with corticosteroid drugs.

Effect of human platelet concentrate, serum and PDGF on RPE cell proliferation and wound healing in vitro

SummaryPurpose: Biological agents like human serum or autologous platelets have recently been employed as adjuvants for macular hole surgery. However, the role of these agents on the retinal cellular level remains unclear. In the present study, we investigated the effect of human platelets, serum and PDGF on RPE migration and proliferation in cell culture. Methods: Human RPE were cultured in DMEM + 2 % FCS and experiments performed at passages 2–4. Human platelet concentrate (PC) and serum (HS) were isolated from blood of patients previewed for macular hole surgery; human PDGF-BB was from Pepro Tech. PC and HS at protein concentrations ranging from 50–1000 μg/ml and PDGF at 1 and 10 ng/ml were added to 5000 cells/well in the proliferation assay and to a confluent RPE monolayer on which a central mechanical “wound” (5 mm diameter) was made. Incubation times ranged from 1 h to 5 days. Cell numbers at D 5 were indirectly determined by protein measurements. In the wound model, the cells inside the wound area were counted and results compared to the control cultures that received no supplements. Results: Cell proliferation was significantly stimulated over controls by all concentrations of PC, HS and PDGF with any incubation time. Compared to PC and PDGF, HS revealed less proliferation after 1–6 h of incubation; there was no significant difference from PC with other incubation times. In the wound model, both PC and PDGF significantly increased the number of cells migrating into the denuded area after 1 h incubation with the culture medium; longer incubation times had no further effect compared to controls. Conclusion: The present study is the first to demonstrate that human platelet concentrate induces proliferation and migration of RPE cells in vitro. However, PDGF, a growth factor which is abundantly present in platelets, was found to be at least equally effective. We assume that the majority of the mitogenic effect of platelet concentrate is due to PDGF.ZusammenfassungHintergrund: Der Effekt des Einsatzes von autologem Thrombozytenkonzentrat (TK) in der Makulachirurgie wurde bisher nur durch klinische Verlaufsbeurteilungen (postoperativer Visus) eruiert. Aufgrund der bekannten Spontanheilungen von Makulalöchern steht der Nachweis einer Wirksamkeit dieser Substanz auf zellulärer Ebene bisher noch aus. In der vorliegenden Studie untersuchten wir den Wundheilungseffekt von TK auf humane RPE-Zellen im Vergleich zu humanem Serum (HS) und dem Wachstumsfaktor PDGF. Material und Methode: Aus Spenderbulbi isolierte RPE-Zellen wurden in DMEM mit 2 % x (FCS) gezüchtet. Autologes TK und HS wurden in Proteinkonzentrationen von 50–1000 μg/ml zum Kulturmedium gegeben (für PDGF: 1 bzw. 10 ng/ml) und für 1 Stunde bis 5 Tage inkubiert. Kontrollkulturen erhielten keinen Zusatz. Nach 5 Tagen erfolgte der Nachweis einer Proliferationsstimulation mittels Zellzählung und Proteinbestimmung. Parallel wurde ein möglicher wundheilungsstimulierender Effekt der für eine Stunde inkubierten Substanzen 5 Tage nach Läsion konfluenter Kulturen über automatisierte Bildanalyse der neu bewachsenen Wundfläche gemessen. Ergebnisse: Im Vergleich zu den Kontrollen führten alle zugesetzten Agentien zu einer signifikanten Proliferationsstimulation mit zunehmender Inkubationszeit und ansteigenden Konzentrationen. TK und PDGF zeigten lediglich bei kürzeren Inkubationszeiten (1–6 h) höhere Proliferationsraten als HS. Auch im Wundmodell konnte durch TK- und PDGF-Zusatz eine im Vergleich zu HS raschere Migration der Zellen gefunden werden. Schlußfolgerung: Erstmalig wird in dieser Studie ein proliferations- und migrationsfördernder Effekt von autologem TK auf humane RPE-Zellen nachgewiesen. Die Wirksamkeit von Thrombozytenkonzentrat zeigt sich bei identischen Konzentrationen der von humanem Serum überlegen; ein vergleichbarer Effekt auf zelluläres Wachstum und Wundheilung läßt sich jedoch auch durch die isolierte Zugabe des Wachstumsfaktors PDGF erzielen. Der wundheilungsfördernde Effekt von TK ist somit möglicherweise durch das in Thrombozyten reichlich vorhandene PDGF bedingt.

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

Abstract 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 (7.5×0.5×0.2 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 short-term 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.

Fibroblast growth factor 2, heparin and suramin reduce epithelial ulcer development in experimental HSV-1 keratitis

Abstract• Background: We have previously shown that basic fibroblast growth factor (FGF-2) enhances corneal epithelial healing in different experimental models in vivo. In order to study the healing effect of this growth factor in pathological conditions of the cornea, we investigated whether topical application of FGF-2 could affect herpes keratitis in rabbits. Since HSV-1 infection is prevented in vitro by incubation with heparin, we also topically applied heparin and suramin, considering the similar interaction of herpes simplex virus and FGF-2 with cell membrane-anchored heparan sulfate. • Methods: After virus inoculation with a human BEY.2 strain, rabbits were treated with either FGF-2 (200 ng to 2 μg/application), heparin (250 μg/application) or suramin (250 μg/application) 4 times daily until day 14. Acyclovir and placebo administrations served as controls (n=48 rabbits). Computerized ulcer surface analysis, clinical observations and virus recovery assays were performed. • Results: Topical FGF-2, heparin and suramin treatment revealed a significant reduction in peak ulcer sizes, and complete epithelial healing was achieved earlier than in placebo-treated corneas. However, no significant antiviral effect of FGF-2, heparin and suramin was detectable in plaque assays from conjunctival swabs. • Conclusions: These experiments demonstrate that FGF-2 is effective in promoting herpetic epithelial ulcer healing, either due to its proliferative effects on epithelial cells or indirectly by occupying the sites on cell surface heparan sulfate necessary for the attachment of the virion. The latter mechanism of action is presumably the reason for the similar effect of heparin and suramin.

Immunreaktion nach perforierender Keratoplastik II. Prävention und Therapie

Zusammenfassung¶· Eine verminderte Sensibilisierung des Empfängers durch reduzierte Antigendifferenz (“MHC-Matching”) oder ¶· eine pharmakologische Behandlung mit Modulation der afferenten und efferenten Immunantwort.

Immunreaktion nach perforierender KeratoplastikII. Prävention und Therapie

Zusammenfassung¶· Eine verminderte Sensibilisierung des Empfängers durch reduzierte Antigendifferenz (“MHC-Matching”) oder ¶· eine pharmakologische Behandlung mit Modulation der afferenten und efferenten Immunantwort.

Immunreaktion nach perforierender Keratoplastik II. Prävention und Therapie: II. Prävention und Therapie

Zusammenfassung¶· Eine verminderte Sensibilisierung des Empfängers durch reduzierte Antigendifferenz (“MHC-Matching”) oder ¶· eine pharmakologische Behandlung mit Modulation der afferenten und efferenten Immunantwort.