Peter Oliver Denk

Evaluation of glaucomatous visual field loss with locally condensed grids using fundus-oriented perimetry (FOP)

PURPOSE We compared detection rates of glaucomatous visual field defects (VFDs) between a conventional rectangular stimulus grid and locally condensed test point arrangements in morphologically suspicious regions. METHODS Humphrey Field Analyzer model 630 (HFA I, program 30-2 with a rectangular 6 x 6 grid) was used as the conventional perimetric method. Individual local test-point condensation was realized by fundus-oriented perimetry (FOP) on the Tuebingen Computer Campimeter (TCC). RESULTS Of a total of 66 glaucoma patients, or suspected sufferers, 23 showed normal findings and 27 showed pathological findings with both methods. In 15 cases we found normal visual fields in HFA 30-2, whereas FOP revealed early glaucomatous functional damage. Only one case showed pathological HFA results, while FOP was normal. Detection rates of VFDs significantly differed between the two methods (p > 0.001; sign test). CONCLUSIONS FOP, using individually condensed test grids, significantly increases detection rates of glaucomatous VFDs in morphologically suspicuous areas compared with a conventional HFA 30-2 technique using equidistant rectangular (6 x 6) test point arrangements. Eur J Ophthalmol 2001; 11 (Suppl 2): S57-S62.

Effect of growth factors on the activation of human Tenon’s capsule fibroblasts

Purpose. To investigate stimulatory effects of PDGF-AA, PDGF-AB, PDGF-BB, bFGF, IL-1ß, TGF-ß1 and TGF-ß2 on the proliferation and myofibroblast transformation of cultured human Tenon’s capsule fibroblasts and to characterize expression of PDGF- and TGF-ß-receptors in these cells. Methods. To determine cell proliferation, cell number of 2nd passage cultured human Tenon’s capsule fibroblasts was measured before and after addition of growth factors using a computer-based cell counter system. Immunoblotting was used to detect and quantitate a-smooth-muscle actin (a-SMA) expression. Expression of PDGF- and TGF-ß-receptor mRNA was detected by RT-PCR, expression of the corresponding protein was demonstrated using Western blot. Results. A significant increase in proliferation (p = 0.05) was detected after exogenous stimulation with PDGF-AA (10ng/ml and 100ng/ml), PDGF-AB (10ng/ml and 100ng/ml), PDGF-BB (10ng/ml and 100ng/ml), bFGF (100 ng/ml), IL-1ß (1 ng/ml and 10 ng/ml), TGF-ß1 (0.5 ng/ml) and TGF-ß2 (0.5 ng/ml). Both TGF-ß1 and TGF-ß2 stimulated expression of a-SMA in a dose dependent manner with peak activity at a concentration of 50 ng/ml (TGF-ß1) and 500 ng/ml (TGF-ß2). Protein and mRNA of PDGF-receptor type a and type ß and TGF-ß-receptors type I, II and III are expressed in cultured human Tenon’s capsule fibroblasts. Conclusions. The present investigation strongly supports the hypothesis that PDGF-isoforms are major stimulators of proliferation of Tenon’s capsule fibroblasts after glaucoma filtering surgery while TGF-ß-isoforms are essential for the transformation of Tenon’s capsule fibroblasts into myofibroblasts.

Proliferative response of cultured human tenon's capsule fibroblasts to platelet-derived growth factor isoforms

Abstract• Background: Although platelet-derived growth factor (PDGF) has been thought to be critical in the wound-healing response of Tenons capsule fibroblasts after glaucoma filtration surgery, no information is currently available concerning the proliferative effect of PDGF isoforms on this cell type. The aim of the present study was to evaluate the proliferative effect of PDGF-AB heterodimer and PDGF-AA and -BB homodimers on cultured human Tenons capsule fibroblasts. • Methods: Human Tenons capsule fibroblasts, cultured under serum-free conditions, were stimulated with PDGF-AA, -AB and -BB isoforms in concentrations ranging from 1 to 100 ng/ml. Cell numbers were determined on days 1, 3, 5 and 7, using a cell counter. • Results: Addition of PDGF-AB and -BB led to a dosedependent increase in cell proliferation. A maximal response (79.9% over control) was obtained after 7 days with 30 ng/ml of PDGF-BB, with an EC50 of 8.9 ng/ml. The maximal increase in cell proliferation caused by PDGF-AB (30 ng/ml) was 54.9%, with an EC50 of 12.5 ng/ml. Stimulation with PDGF-AA revealed a significant effect only with concentrations higher than 30 ng/ml. • Conclusion: Our results indicate that PDGF-AB and -BB isoforms are potent stimulators of proliferation of human Tenons capsule fibroblasts, suggesting that PDGF-AB and -BB isoforms play an important role in the wound-healing response after glaucoma filtration surgery.

The in vitro effect of platelet-derived growth factor isoforms on the proliferation of bovine corneal stromal fibroblasts depends on cell density

Abstract• Purpose: Recently, it has been shown that corneal stromal fibroblasts express the mRNA for PDGF-β-type receptors, while corneal epithelial cells express the mRNA for the PDGF B-chain, suggesting a role of PDGF isoforms in the regulation of corneal homeostasis and wound healing via an unidirectional epithelial to stromal paracrine interaction. The purpose of this study was to characterize the proliferative response of cultured bovine corneal stromal fibroblasts to PDGF isoforms. • Methods: Bovine corneal stromal fibroblasts were seeded at a cell density of 60 cells/mm2 (low density) and 120 cells/mm2 (high density) and were cultured under serum-free conditions. Except for corresponding controls, PDGF AA, BB and AB (obtained by separate expression of cloned genes inE. coli) were added in concentrations ranging from 3.125 to 100 ng/ml. Cell numbers were determined after an incubation period of 6 days using a cell counter. • Results: Stromal fibroblasts, when cultured at a high density, revealed constant cell numbers during the whole incubation period. Under these culture conditions, stimulation with PDGF AA, BB and AB led to a significant dose-dependent increase in cell proliferation. When cultured at a low cell density, stromal fibroblasts revealed a significant reduction of cell numbers after 6 days of incubation. This reduction was prevented by PDGF AA and AB isoforms in a dose-dependent manner. In contrast, PDGF BB was not effective. • Conclusion: The results of the “high-density” assays suggest that PDGF isoforms act as mitogens for stromal fibroblasts during wound healing, when density of fibroblasts is high. The results of the “low-density” assays support the idea that PDGF AA and AB can prevent cell loss during corneal homeostasis when density of keratocytes is low.

Effect of heparin on human corneal fibroblast proliferation in vitro with and without growth factor stimulation

Abstract · Background: The outcome of keratorefractive procedures such as PRK and LASIK is limited by the wound-healing process in the corneal stroma, which gives rise to complications such as haze formation and regression. The proliferation and matrix synthesis of corneal stromal fibroblasts is the central element of the wound-healing process. In order to develop new therapeutic strategies to reduce wound-healing intensity, we investigated the effect of heparin on the proliferation of cultured human corneal stromal fibroblasts (HCF) alone and in the presence of growth factors. · Methods: Primary cultures of HCF were established using epithelium and endothelium-free explants. Secondary cultures of HCF (first passage), cultured in WM/F12 supplemented with 10 µg/ml transferrin and 10 µg/ml thyroglobulin (LR-1 medium), 1% fetal calf serum (FCS) and 10% FCS were used to determine the effect of heparin on the proliferation of HCF in concentrations ranging from 12.5 µg/ml to 5000 µg/ml. Cell number was determined using the CASY 1 cell counter system. Modulation of HCF proliferation by heparin (50 µg/ml and 2000 µg/ml) was also investigated under serum-free conditions and in the presence of bFGF, EGF and PDGF-BB. · Results: Addition of heparin led to a dose-dependent inhibition of proliferation after 6 days of incubation, which was statistically significant for 500–5000 µg heparin/ml (FCS 1%) and for 200–5000 µg heparin/ml (FCS 10%). IC50 values for this effect were determined to be approximately 700 µg heparin/ml. When cultured under serum-free conditions (LR-1), a significant reduction of cell number was only observed with 5000 µg heparin/ml. There was no significant modulation of PDGF-BB-, bFGF-, or EGF-stimulated cell proliferation by heparin at concentrations of 50 µg/ml and 2000 µg/ml after 6 days of incubation. · Conclusion: Our observations indicate that heparin can inhibit proliferation of HCF effectively. The results of the present study could eventually pave the way to prevent anterior stromal haze formation and regression after keratorefractive surgery.

Sind filtrierende Eingriffe bei Glaukompatienten mit ausgedehnten Gesichtsfeldausfällen mit einem größeren funktionellen Risiko verbunden

ZusammenfassungHintergrund: Untersucht wurde die Inzidenz eines Visusverlustes durch einen Ausfall des zentralen Gesichtsfeldes und der foveolaren Fixation in der ersten Woche nach einer filtrierenden Glaukomoperation. Patienten: In die Studie wurden 408 Patienten eingeschlossen, bei denen von Januar 1993 bis April 1997 an der Unversitätsaugenklinik Tübingen eine filtrierende Glaukomoperation durchgeführt wurde und deren Verlauf bis 12 Monate (± 3 Monate) postoperativ bekannt war. Die Auswertung der präoperativen, intraoperativen und postoperativen Daten erfolgte retrospektiv. Ausgeschlossen wurden alle Patienten, bei denen keine ausreichende Verlaufskontrolle durchgeführt werden konnte, bei denen gleichzeitig eine Kataraktextraktion durchgeführt und bei denen weitergehende operative Eingriffe (z.B. Moltenoimplantat) eingesetzt wurden. Ergebnisse: Bei 404 Patienten (99,3%) fand sich kein Visusverlustes durch einen Ausfall des zentralen Gesichtsfeldes und der foveolaren Fixation in der ersten Woche nach einer filtrierenden Glaukomoperation. Von diesen Patienten wiesen 11 eine deutliche postoperative Visusreduktion (>2 dB) auf, die durch eine Zunahme der Linsentrübung erklärt war, bei einem Patienten zeigte sich eine progressive altersbedingte Makulopathie. Bei einem Patienten (0,2%) fand sich ein progredienter relativer Gesichtsfeldausfall im zentralen Bereich. Bei 2 Patienten (0,5%) ließ sich bereits unmittelbar nach der Operation ein Verlust der Fixation und ein Verlust des zentralen Gesichtsfeldes nachweisen. Schlussfolgerung: Ein Verlust der Sehschärfe durch Einbruch des zentralen Gesichtsfeldes und der zentralen Fixation unmittelbar nach einer filtrierenen Operation ist eine seltene Komplikation. Selbst fortgeschrittene Gesichtsfeldausfälle sind daher keine Kontraindikation für eine filtrierende Glaukomoperation.SummaryBackground: We evaluated the prevalence of the loss of visual acuity due to loss of the central portion of the visual field and foveolar fixation in the first week after glaucoma filtering surgery. Patients and methods: We included 408 patients, in whom glaucoma filtering surgery was performed between January 1993 and April 1997 at the University Eye Clinic in Tübingen and who had completed 1-year follow-up examinations. The retrospective evaluation included preoperative, intraoperative and postoperative data. We excluded all patients who did not complete 1-year follow-up examinations (12±3 months), who have died during the 1-year follow-up, who had combined glaucoma and cataract surgery or in whom the Molteno implant procedure was performed. Results: A total of 404 patients (99.3%) did not suffer loss of the central visual field and foveolar fixation in the first week after glaucoma filtering surgery. In 11 cases, loss of visual acuity >2 dB was due to progressive lens opacification. One patient suffered from postoperative progression of his age-related maculopathy. In one patient (0.2%) progression of a preexisting relative central scotoma occurred immediately after the operation. Two patients (0.5%) suffered from loss of fixation and the central visual field immediately after glaucoma filtering surgery. Conclusions: Loss of the central visual field and central fixation immediately after glaucoma filtering surgery is a rare complication. Therefore, glaucoma filtering surgery can also be recommended for patients with advanced visual field defects.

Inhibitory effect of Trapidil on the proliferation of bovine corneal fibroblasts in vitro

Abstract · Background: In order to develop new strategies for the pharmacological modulation of posttraumatic and postsurgical wound healing of the corneal stroma, the effect of Trapidil, a competitive platelet-derived growth factor (PDGF) antagonist, on the proliferation of cultured bovine stromal fibroblasts (BSF) was investigated. · Methods: BSF, obtained from explant cultures, were seeded at a cell density of 100/mm2. The effect of various concentrations of Trapidil on cell viability and cell proliferation was determined using three different culture conditions: (1) serum-free medium (WM/F12), (2) serum-containing medium (WM/F12+10% FCS), and (3) serum-free medium +50 ng/ml PDGF-BB. Trapidil was added in concentrations ranging from 100 μg/ml to 400 μg/ml. Cell numbers were determined 2 and 5 days after addition of Trapidil, using a computer-based cell-counting system. Cell viability was evaluated morphologically and by means of a repopulation assay. · Results: Addition of Trapidil (100–400 μg/ml) led to a significant, dose-dependent inhibition of both serum- and PDGF-BB-induced proliferation of BSF. In contrast, treatment of quiescent BSF, cultured in serum-free medium, did not result in a significant reduction of cell number. No cytotoxic effects were observed. · Conclusion: The results of the present study demonstrate an inhibitory effect of Trapidil on the proliferation of BSF. It can be assumed that application of Trapidil might be a useful tool in the prevention of corneal complications after trauma (e.g., scarring, astigmatism and – with respect to photorefractive procedures – formation of haze and regression of the refractive effect).

Neue klinische und epidemiologische Aspekte von Episkleritis und Skleritis

Skleritis und Episkleritis können im Rahmen zahlreicher Systemerkrankungen auftreten. In bestimmten Fällen können subjektive Symptome und visusbedrohende Komplikationen langfristig nur durch eine systemische Immunsuppression angegangen werden. Therapeutische Leitlinien zur Immunsuppression bei Episkleritis- und Skleritispatienten fehlen bis heute.Patienten und Methode: In einer retrospektiven Studie wird analysiert, welche systemischen Erkrankungen unserer Patientenpopulation zugrundeliegen und zu welchem Zeitpunkt sie manifest wurden. Es soll überprüft werden, ob eine Skleritis und eine Episkleritis nacheinander am gleichen Auge aufgetreten sind. Weiterhin wird untersucht, ob die Diagnose einer systemischen Erkrankung einen Hinweis auf die Notwendigkeit einer längerfristigen systemischen nichtsteroidalen Immunsuppression gibt.Ergebnisse: Zwischen 1991 und 1995 wurden in der Universitäts-Augenklinik Tübingen 68 Patienten mit entzündlichen Skleraerkrankungen behandelt. Bei 13 Patienten war eine systemische Grunderkrankung vor Beginn der Augenbeschwerden bekannt, bei 8 Patienten wurde sie im weiteren Verlauf diagnostiziert. Weder bei den Episkleritis- noch bei den Skleritispatienten bestand ein statistisch signifikanter Zusammenhang zwischen der Diagnose einer assoziierten Systemerkrankung und der Notwendigkeit einer Therapie mit nichtsteroidalen Immunsuppressiva. Bei nur 2 Patienten (3%) wechselte ein Krankheitsbild in das andere über.Schlußfolgerung: Die geringe Zahl der Übergänge zwischen Episkleritis und Skleritis spricht dafür, daß es sich bei den beiden Krankheitsbildern um 2 eigenständige Entitäten handelt, die möglicherweise durch verschiedene Mechanismen hervorgerufen werden. Die Therapie mit Immunsuppressiva sollte nach unseren Ergebnissen nicht allein von einer Systemerkrankung, sondern vom okularen Befund abhängig gemacht werden.Numerous systemic diseases can cause scleritis or episcleritis. Frequently, symptoms and complications compromising vision can only be managed with systemic immunosuppressants. There are no clear guidelines on the indications for systemic immunosuppressants in patients with episcleritis and scleritis.Patients and methods. The aim of the present retrospective study was to investigate how many patients with episcleritis or scleritis have an associated systemic disease and at what stage it is diagnosed. Secondly, the proportion of patients who present with episcleritis or scleritis in the first instance and then change into the other category during the course of the disease was analyzed. Finally, we checked whether the presence of an associated systemic disease indicates the necessity to treat the patient with nonsteroidal systemic immunosuppressive drugs.Results. Sixty-eight patients with inflammatory scleral diseases were treated at the University Eye Clinic between 1991 and 1995. In 13 patients an associated systemic disease was diagnosed before the appearance of ocular symptoms, and in 8 patients such an illness was diagnosed at a later stage. In 2 cases (3%) the ocular disease category changed during the course of the disease. Neither in the episcleritis nor in the scleritis population was a statistically significant correlation established between the diagnosis of an associated systemic disease and the necessity to treat the patient with nonsteroidal systemic immunosuppressive drugs.Conclusion. The small number of patients who changed the ocular disease category may indicate that episcleritis and scleritis are two independent entities, which might even be caused by different mechanisms. The indications for the management of episcleritis and scleritis with immunosuppressive drugs should not only depend on the diagnosis of an associated systemic disease, but also and mainly on the severity of the ocular manifestation.

Serum-free cultivation of bovine stromal fibroblasts

SummaryThe purpose of the present study was to conduct a comparative evaluation of the effect of several serum-free culture conditions on adhesion, population doubling, cryopreservation and PDGF-induced effects on cell proliferation of bovine stromal fibroblasts (BSF). Additionally, these effects were compared to serum-containing cultures. Methods: Only second-passage BSF were used. Cells were cultured using four different culture media (WM/F12, WM/F12 + FCS 1 %, LR-1, DMEM). After 24 h, plating efficiency was determined using a cell-counter system. Subsequently, the cells were seeded at a density of 100 cells/mm2 and cultured for 10 days using the different culture media. Cell number was determined at day 2, 4, 7 and 10 after seeding. Furthermore, the effect of 50 ng/ml PDGF-BB on the proliferation of BSF was tested for these conditions. Cell vitality was determined after cryopreservation of two weeks for each culture medium. Results: The plating efficiency of BSF ranged from 50.2 to 55.5 % for the serum-free culture media in contrast to serum-containing conditions, where plating efficiency was 94.8 %. With WM/F12 + FCS 1 %, a population doubling of 1.27 was observed after an incubation period of 10 days. In contrast, cultivation under serum-free conditions caused neither significant cell proliferation nor cell loss. The stimulation of cell proliferation with PDGF-BB was shown to be 28 % (LR1), 40 % (WM/F12 + FCS 1 %), 76 % (WM/F12) and 95 % (DMEM) compared to the control. While cell vitality after cryopreservation was found to be 62.7 % using WM/F12 + FCS 1 %, cell vitality using serum-free media was 12.6–22.8 %. Conclusions: The results of the present study demonstrate that with respect to optimal cell adhesion and cell vitality after cryopreservation, serum-containing media should be used. BSF cultured under the serum-free conditions used in the present study can be maintained quiescent and vital for at least 10 days. Therefore, these serum-free media are useful for cell-culture studies (e. g., determination of proliferation and cytotoxicity).ZusammenfassungHintergrund: Untersucht wurde der Einfluß verschiedener serumfreier Kulturmedien im Vergleich zu serumhaltigem Kontrollmedium auf Adhäsion, Proliferation, Populationsdynamik und Kryolagerung boviner stromaler Fibroblasten (BSF) sowie auf den proliferationssteigernden Effekt eines Referenzzytokins (PDGF-BB). Methode: Für die Versuche wurden ausschließlich BSF der 2. Passage verwendet. Zunächst wurden die Zellen in 4 verschiedenen Medien (WM/F12, WM/F12 + FCS 1 %, LR-1, DMEM) ausgesät. Nach 24 h wurde die Adhäsionseffizienz mit einem Zell-Counter-System ermittelt. Anschließend wurden BSF bei einer Zelldichte von 100 Zellen/mm2über 10 Tage in den genannten Medien kultiviert. Am 2., 4., 7. und 10. Tag nach der Aussaat wurde jeweils die Zellzahl bestimmt. Weiterhin wurde der Effekt von 50 ng/ml PDGF-BB als Referenzzytokin auf die Proliferation von BSF unter diesen Bedingungen ermittelt. Schließlich wurde die Zellvitalität nach 2wöchiger Kryolagerung in flüssigem Stickstoff für die 4 Medien bestimmt. Ergebnisse: Die Adhäsionseffizienz von BSF lag in serumfreien Medien zwischen 50,2 und 55,5 %, in WM/F12 + FCS 1 % bei 94,8 %. Die Untersuchung des Einflusses von WM/F12 + FCS 1 % auf die Zellproliferation ergab eine Zellverdopplungsrate von 1,27 nach 10 Tagen. Nach 10tägiger Kultivierung in den serumfreien Medien ergab sich keine signifikante Zellproliferation bzw. Zellverlust. Die Steigerung der Zellproliferation mit PDGF-BB im Vergleich zum Kontrollmedium lag nach 6 Tagen Inkubation mit LR-1 bei 28 %, mit WM/F12 + FCS 1 % bei 40 %, mit WM/F12 bei 76 % und mit DMEM bei 95 %. Während die Zahl vitaler Zellen nach Kryolagerung mit WM/F12 + FCS 1 % bei 62,7 % lag, wurde für die serumfreien Medien lediglich ein Anteil von 12,6–22,8 % vitaler Zellen ermittelt. Schlußfolgerung: Die Ergebnisse dieser Studie zeigen, daß serumhaltiges Medium insbesondere für die Zelladhäsion und Kryolagerung verwendet werden sollte. Alle getesteten serumfreien Medien eignen sich potentiell für die Analyse von Zytokineffekten bzw. Zytotoxizitätstestungen, da BSF für mindestens 10 Tage vital und in einem proliferativen Ruhezustand gehalten werden können.

Effektivität von Dorzolamid als Zusatztherapie bei Glaukompatienten mit maximal tolerierter medikamentöser Therapie. Eine Pilotstudie

Background The purpose of this study was to evaluate prospectively the value of dorzolamid as an adjunct to maximum tolerated medical therapy in glaucoma patients in whom surgery would otherwise be required. Methods 32 eyes of 21 patients with primary open angle glaucoma (14 patients), glaucoma in aphakia (3 patients), pigmentary glaucoma (2 patients), juvenile glaucoma (1 patient) and exfoliative glaucoma (1 patient) were included. The effect of additional dorzolamid application on intraocular pressure (IOP) was determined after 2 hours, 4 days, 4 weeks, 3 months and 6 months. After 6 months, visual fields were checked. Results Average reduction of IOP in eyes in which dorzolamid was continued was determined to be 31% after 2 hours, 18.3% after 4 days, 17.9% after 4 weeks, 12.1% after 3 months and 9.7% after 6 months. 19 (59%) eyes continued to receive dorzolamid after 6 months without being operated. No progression of glaucomatous damage could be detected in these eyes. In 11 (34%) eyes, treatment with dorzolamid was discontinued. 2 patients (4 (12%) eyes) did not tolerate local side effects of dorzolamid. In 7 (22%) eyes reduction of IOP was insufficient and filtration surgery had to be performed immediately. Conclusion The results of the present study demonstrate that dorzolamid represents an alternative to an immediate surgical management in patients on maximum tolerated therapy for at least six months.