[Glaucoma and oxidative stress. Determination of malondialdehyde--a product of lipid peroxidation]. / Glaukom und oxidativer Stress. Bestimmung von Malondialdehyd--einem Lipidperoxidationsprodukt.

BACKGROUND: Primary open-angle glaucoma (POAG) is a multifactorial disease, in which aging, race, genetic disposition, vascular status, probably inflammation, and maybe oxidative stress may play a specific role. We wanted to know if by using a common test for a specific oxidative stress product differences would be found in the aqueous humor and serum of patients with POAG, cataract without pseudoexfoliation, cataract with pseudoexfoliation, and volunteers without ocular disease. PATIENTS AND METHODS: We examined the aqueous humor of 33 patients with POAG, 111 patients with cataract without pseudoexfoliation, 39 patients with cataract and pseudoexfoliation syndrome and the serum of all three groups plus of 43 volunteers without ocular disease. Malondialdehyde as an oxidative stress product of peroxidation of lipids was proven by thiobarbituric acid-reacting substances (TBARS). RESULTS: The amount of TBARS is given in micromol/l: (1) in the serum of the group with cataract 1.176, with cataract and pseudoexfoliation 1.019, with POAG 0.992, and with healthy eyes 0.983; (2) in the aqueous humor of the group with cataract 0.279, with cataract and pseudoexfoliation 0.274, and with POAG 0.298. There were no statistically significant differences of TBARS (p<0.05) in either the aqueous humor or in the serum of patients with POAG in comparison to those patients without POAG. However, there was a significantly positive correlation between the values in the serum and the aqueous humor. CONCLUSION: For the first time malondialdehyde as a product of lipid peroxidation was determined in the aqueous humor of glaucomatous eyes. With the TBARS method used in our study, it was not possible to detect statistically significant differences of the lipid peroxidation product malondialdehyde between patients with or without POAG. It has to be taken into account that the cataract of the patients in the control group might be a disease per se caused by at least a certain amount of oxidative stress and that these subjects might therefore not be ideal as a control group.

TNF-alpha promoter polymorphisms and primary open-angle glaucoma.

PURPOSE: Primary open-angle glaucoma (POAG) is a multifactorial optic neuropathy with a strong hereditary component. Recent studies suggested a role for tumour necrosis factor-alpha(TNF-alpha) in the pathogenesis of POAG. The purpose of the present study was to investigate a hypothesized association between the TNF-alpha-308G>A and -238G>A gene polymorphisms and the presence of POAG in a Caucasian population. METHODS: The present case-control study comprised 114 unrelated patients with POAG and 228 healthy control subjects, matched for age and gender. Genotyping of the TNF-alpha-308G>A and -238G>A polymorphisms was performed using polymerase chain reaction. RESULTS: Allelic frequencies and genotype distributions of both the TNF-alpha-308G>A and -238G>A gene polymorphisms did not significantly differ between patients with POAG and control subjects. Presence of the TNF-alpha-308A-allele was associated with an odds ratio (OR) of 0.96 for POAG, whereas an OR of 0.52 was found among carriers of the TNF-alpha-238A-allele. CONCLUSION: Our data suggest that none of the investigated TNF-alphagene polymorphisms is a major risk factor among Caucasian patients with POAG.

Iodide iontophoresis as a treatment for dry eye syndrome.

BACKGROUND/AIMS: Among the causes related to the development or perpetuation and aggravation of dry eye disease, oxidative reactions may have a role in the pathogenesis of this disorder. Antioxidants, such as iodide, have shown a strong effect in preventing the oxidative damage to constituents of the anterior part of the eye. In this clinical trial the effectiveness of iodide iontophoresis and iodide application without current in moderate to severe dry eye patients was compared. METHODS: 16 patients were treated with iodide iontophoresis and 12 patients with iodide application without current for 10 days. Subjective improvement, frequency of artificial tear application, tear function parameters (break up time, Schirmer test without local anaesthesia), vital staining (fluorescein and rose bengal staining) as well as impression cytology of the bulbar conjunctiva were evaluated before treatment, 1 week, 1 month, and 3 months after treatment. RESULTS: A reduction in subjective symptoms, frequency of artificial tear substitute application, and an improvement in certain tear film and ocular surface factors could be observed in both groups. A stronger positive influence was seen after application of iodide with current (iontophoresis), as observed in a distinct improvement in break up time, fluorescein and rose bengal staining, and in a longer duration of this effect compared with the non-current group. No significant change in Schirmer test results and impression cytology were observed in both groups. CONCLUSIONS: Iodide iontophoresis has been demonstrated to be a safe and well tolerated method of improving subjective and objective dry eye factors in patients with ocular surface disease.

Cytotoxicity evaluation of soft contact lens care solutions on human conjunctival fibroblasts.

PURPOSE: To determine whether different contact lens care solutions for soft lenses cause damage to human conjunctival cells. METHODS: Primary cultured human conjunctival fibroblasts were incubated with various concentrations of four different commercially available soft contact lens care solutions (OptiFree, Renu, SoloCare, Titmus) at concentrations of 5, 10 and 50 microl/ml medium. Toxicity was examined by determination of (1) the cell viability and mitochondrial activity with the colorimetric MTT test, and (2) the number of living cells with a cell analysis system (CASY 1) as compared with untreated cells. RESULTS: For all four soft contact lens care solutions at a concentration of 5 mul/ml medium, no significant decrease in mitochondrial activity of the human conjunctival fibroblasts was found by the MTT test. At 10 microl/ml, only OptiFree and Titmus reduced mitochondrial viability significantly. The greatest reduction in mitochondrial activity occurred with all of the four soft contact lens care solutions at a concentration of 50 microl/ml. No significant decrease in the number of living conjunctival fibroblasts was observed by CASY 1 even at higher concentrations of the four solutions investigated. CONCLUSION: This in vitro study demonstrates that the examined soft contact lens care solutions induce changes in mitochondria of human conjunctival cells only at higher doses as observed by the MTT test. However, this damage to the mitochondria did not lead to cell death as shown by the cell analysis system.

Role of factor V Leiden and prothrombin 20210A in patients with retinal artery occlusion.

PURPOSE: Retinal artery occlusion is a common vision-threatening disease. Among other risk factors, coagulopathies leading to a hypercoagulable state have been associated with retinal artery occlusion. Numerous studies have shown that two genetic variants, factor V Leiden and prothrombin 20210A, cause a procoagulant state. However, their role in the pathogenesis of retinal artery occlusion is still unclear. The purpose of the present study was therefore to investigate a possible association between factor V Leiden, prothrombin 20210A, and retinal artery occlusion. METHODS: In the present retrospective case-control study, we studied 136 patients with retinal artery occlusion and 136 age- and gender-matched control subjects. The presence of factor V Leiden and prothrombin 20210A alleles was determined by polymerase chain reaction. RESULTS: The prevalence of heterozygosity for the prothrombin G20210A variant did not significantly differ between patients and controls (three patients vs two controls, P=0.65). Distribution of factor V Leiden genotypes revealed no significant difference among the two groups (heterozygosity: eight patients vs 11 controls, P=0.47). As for other risk factors, arterial hypertension, a history of stroke and myocardial infarction were significantly more frequent in patients than in controls. CONCLUSION: Our data suggest that factor V Leiden and prothrombin 20210A do not play a major role in patients with retinal artery occlusion.

Botulinum toxin A treatment in patients suffering from blepharospasm and dry eye.

BACKGROUND: Many patients with essential blepharospasm also show dry eye signs and symptoms. Botulinum toxin A is an effective treatment for reducing spasms in these patients. In this investigation, the effect of botulinum toxin A injections on tear function and on the morphology of the ocular surface in patients suffering from blepharospasm in combination with a dry eye syndrome was investigated. METHODS: Botulinum toxin A injections were applied to 16 patients with blepharospasm. All patients complained of dry eye symptoms and had reduced tear break up time values. A subjective questionnaire and ocular examinations including tear break up time, Schirmer test without local anaesthesia, and rose bengal staining were evaluated before, 1 week, 1 month, and 3 months after injection. Impression cytology was performed before, 1 month, and 3 months after botulinum toxin A treatment. RESULTS: Although all patients were relieved of blepharospasm after botulinum toxin injections, only three noticed an improvement in dry eye symptoms. Eight patients noticed no difference and five complained of worsening. Tear break up time was found to be increased 1 week and 1 month after injections. Schirmer test measurements were reduced up to 3 months. Rose bengal staining slightly increased 1 week after injections. Impression cytology showed no definite change in conjunctival cell morphology 1 month and 3 months after botulinum toxin A injections. CONCLUSION: In the patients presented here suffering from blepharospasm and dry eye, botulinum toxin A injections were effective in relieving blepharospasm but were not successful in treating dry eye syndrome.

Role of factor XIII Val34Leu polymorphism in retinal artery occlusion.

BACKGROUND AND PURPOSE: Factor XIII (FXIII) Val34Leu, a common polymorphism in the gene for factor XIII, has been associated with a lower risk of stroke, myocardial infarction, and deep vein thrombosis. Ineffective fibrin cross-linking has been suggested to be causative. The aim of the present case-control study was to investigate the role of FXIII Val34Leu polymorphism in patients with retinal artery occlusion. METHODS: A total of 108 patients with retinal artery occlusion and 313 age- and sex-matched controls were genotyped for the FXIII Val34Leu polymorphism. Factor XIII Val34Leu genotypes were determined by use of allele-specific polymerase chain reaction. RESULTS: Homozygous Leu genotype was found significantly more often in control subjects than in patients with retinal artery occlusion (P=0.018), with an odds ratio of 0.22 (95% confidence interval 0.07 to 0.74). Distribution of the Val/Val and Val/Leu genotypes did not differ significantly between groups. CONCLUSIONS: Because prevalence of homozygous Leu genotype was significantly higher in controls, we conclude that the Leu/Leu genotype is associated with a protective effect against retinal artery occlusion.

Hyperhomocyst(e)inaemia, but not MTHFR C677T mutation, as a risk factor for non-arteritic ischaemic optic neuropathy.

BACKGROUND/AIMS: Hyperhomocyst(e)inaemia has been identified as a strong risk factor for stroke, myocardial infarction, and deep vein thrombosis. A point mutation of methylene tetrahydrofolate reductase (MTHFR C677T) has been associated with increased plasma homocyst(e)ine levels. To investigate whether hyperhomocyst(e)inaemia and/or MTHFR C677T mutation are associated with non-arteritic ischaemic optic neuropathy (NAION), a case-control study including 59 consecutive patients with NAION and 59 controls matched for age and sex was performed. METHODS: Fasting plasma homocyst(e)ine levels, MTHFR C677T genotypes, and plasma levels of folate and vitamin B-12 were determined. RESULTS: Mean plasma homocyst(e)ine levels were significantly higher in patients than in controls (11.8 (SD 5.7) micromol/l v 9.8 (2.5) micromol/l, p = 0.02). The odds ratio for patients with homocyst(e)ine levels exceeding the 95th percentile of control homocyst(e)ine levels was 5.8 (95% CI 1.5-21.4). Mean plasma folate levels were significantly lower in patients than in controls (4.3 (1.7) ng/ml v 5.5 (1.9) ng/ml, p = 0.001), whereas plasma vitamin B-12 levels did not differ significantly. Prevalence of the MTHFR C677T mutation was not significantly increased in patients with NAION compared with controls. CONCLUSION: These results suggest that hyperhomocyst(e)inaemia, but not MTHFR C677T mutation is associated with NAION. Determination of plasma homocyst(e)ine levels might be of diagnostic value in patients with NAION.

Hyperglycaemia in vitro alters the proliferation and mitochondrial activity of the choriocarcinoma cell lines BeWo, JAR and JEG-3 as models for human first-trimester trophoblast.

AIMS/HYPOTHESIS: Early intrauterine growth delay in diabetes could be caused by a reduced growth of the placenta. Our study investigates whether hyperglycaemia in vitro reduces trophoblast proliferation. METHODS: First-trimester trophoblast cell models (BeWo, JAR and JEG-3 choriocarcinoma cells) were cultured for 24 and 48 h with 5.5 mmol/l D-glucose, 25 mmol/1 D-glucose (hyperglycaemia) and with an osmotic control. Cell number, total protein and nucleic acid content and mitochondrial activity (tetrazolium salt assay) were measured, the cell cycle analysed (FACS, cyclin B1 levels) and apoptosis (Annexin-V) measured. RESULTS: In BeWo cells hyperglycaemia reduced cell number, protein, nucleic acid and cyclin B1 levels. The reduced G2/M and increased G0/G1 population after 24 h reflects growth arrest at G0/G1. In JAR cells after 24 h the population was arrested in G0/G1, whereas after 48 h the G0/G1 block was abrogated and the cells were arrested at G2/M. The net effect was an unchanged cell number. In JEG-3 cells hyperglycaemia resulted in fewer cells after 24 h but not after 48 h indicating some adaptation. Mitochondrial activity was either stimulated (BeWo) or reduced (JAR, JEG-3) under hyperglycaemia. Some of these effects were also induced by hyperosmolarity alone. CONCLUSION/INTERPRETATION: Hyperglycaemia has the potential to inhibit the proliferation of first-trimester trophoblast cell models. The mechanisms leading to growth arrest and to changes in mitochondrial activity are complex and depend on differentiation. We hypothesise a hyperglycaemia-induced impairment of placental growth in the first trimester of a poorly controlled diabetic pregnancy.

Ocular Ferning test - effect of temperature and humidity on tear Ferning patterns.

BACKGROUND: The ocular ferning test is used as a diagnostic aid to evaluate patients with dry-eye disease. The ferning phenomenon is a dendritic growth form of dried tear fluid. The influence of temperature and relative humidity (rH) on the morphology of tear ferning patterns was investigated. METHODS: Two microliters of tear fluid of 5 volunteers with normal tear function were dropped onto a microscope slide with a pipette and were dried in a climatic chamber at four different temperatures (20, 22, 24 and 26 degrees C) and at four different grades of rH (30, 40, 50 and 60%) at each temperature. The dried droplet was divided according to distinct morphological features into a center, transition zone and margin. The center of the dried droplet was classified into four types according to the system of Rolando. RESULTS: In the temperature range between 20 and 26 degrees C, rH has a strong influence on the ferning patterns. At these temperatures, increasing rH resulted in a deterioration of type I ferning obtained at 30% rH to type II or type III at 60% rH in all subjects. Otherwise, at rH of 30, 40 and 50%, no definite influence of temperature between 20 and 26 degrees C was observed. At an rH of 60%, the ferning patterns changed with increasing temperature from 20 to 26 degrees C from type II to type III. CONCLUSION: High humidity can modify and deteriorate the ferning patterns of tear fluid from subjects with normal tear function. To obtain reproducible results using the ocular ferning test, stable conditions are necessary. An rH not higher than 50% at a temperature range between 20 and 26 degrees C produced ferning patterns without interstitial spaces which could be related to the best quality according to the system of Rolando.

[Quantifying the damage to conjunctival and corneal cell cultures caused by uv light using CASY (Cell Analysis System). A method for reducing animal experiments]. / Quantifizierung der Schädigung von Bindehaut- und Hornhautzellkulturen durch UV-Licht mit Hilfe des CASY (Zellanalyse)-Systems. Eine Methode zur Reduktion von Tierversuchen.

BACKGROUND: By depletion of stratospheric ozone, enhanced levels of UV radiation reach the surface of the earth. Exposure of the anterior parts of the eye to UV radiation leads to irritation of the conjunctival and corneal cells. METHOD: By the CASY (cell analysis) system the influence of UV radiation on cultures of conjunctival and corneal cells was observed by determination of the cell counts, cell diameter, and the cell volume. RESULTS: By comparing these parameters with the control, damage of the conjunctival and corneal cells by UV radiation can be determined within 2 s of exposure of the cells in quartz glass vials to the UV light. CONCLUSION: By the CASY system the dramatic influence of UV radiation on cells of the anterior parts of the eye can be determined. This system enables objective statements on the cytotoxicity of radiation, chemical substances and drugs on cell cultures without the use of radioactive methods, complicated determination of cell metabolism or staining methods which are difficult to standardize. Also, studies on animals can be reduced by the CASY system.

In vitro interactions of oxidatively modified LDL with type I, II, III, IV, and V collagen, laminin, fibronectin, and poly-D-lysine.

The accumulation of LDL in the arterial intima is considered a key event in atherogenesis. We investigated the binding of oxidized LDL (ox-LDL) to microtiter plates coated with type I or II collagen, laminin, fibronectin, or poly-D-lysine. Oxidation of LDL, 125I-LDL, or Eu(3+)-LDL was performed with CuCl2, varying the time of oxidation. Bound lipoprotein was assessed by counting radioactivity or fluorescence in the wells. Binding of highly ox-LDL in PBS followed the order: type I collagen > poly-D-lysine > type II collagen > laminin > fibronectin. Comparing various collagen types, the binding of ox-LDL followed the order: type I > type V and, type III > type IV > type II collagen. Binding of ox-LDL in PBS was dependent on an increase in negative charge of ox-LDL. Testing certain amino acids as competitors for binding of highly ox-LDL to type I collagen put lysine first, followed by arginine and histidine. On laminin, histidine competed most, followed by lysine and arginine. When studying the influence of Na+, K+, Ca2+, Mg2+ (equivalent to their concentrations in the interstitial fluid), native LDL, moderately ox-LDL, and highly ox-LDL showed the same affinity to type I collagen. However, a fivefold dilution of the buffer increased the affinity of moderately and highly ox-LDL 3.9- and 10-fold compared with native LDL. Application of the F(ab')2 from a monoclonal antibody to ox-LDL revealed a strong competition of the binding of highly ox-LDL to type II collagen (60%), laminin (35%), type I collagen (20%), and poly-D-lysine (15%), whereas the binding to fibronectin was not affected.

[Effect of calcium dobesilate on progression of diabetic retinopathy]. / Einfluss von Kalziumdobesilat auf die Progression der diabetischen Retinopathie.

BACKGROUND: The increase of platelet aggregation, plasma and whole blood viscosity and the decrease of red cell deformability in patients with diabetes mellitus may be causative in the development of retinopathy. A therapy influencing these factors maybe prevents retinopathy or slows progression. MATERIAL AND METHODS: In a clinical and prospective study over 2 years 74 patients aged 20 to 69 years with a mild background retinopathy were randomly assigned to take oral 3 x 500 mg calcium dobesilate (Doxium) or no therapy. Both groups did neither differ in demographic nor in laboratory data. In fluorescein angiography following characteristics of retinopathy were graded: microaneurysms, size and outline of foveal avascular zone, capillary loss and severity of dye leakage. Additionally in 15 patients contrast sensitivity, visual fields and oscillatory potentials were investigated. RESULTS: The results of the fluorescein angiographical characteristics revealed no statistically significant difference between both groups. In the oscillatory potentials a significant shortening of latency could be found in both groups, but there was no difference between the groups. CONCLUSION: A 2-years application of calcium dobesilate has no statistically significant influence on the progression of diabetic retinopathy.

[Non-arteritic anterior ischemic optic neuropathy: long-term results after hemodilution therapy]. / Nichtarteriitische vordere ischämische Optikusneuropathie: Langzeitergebnisse nach Hämodilutionstherapie.

BACKGROUND: In patients with nonarteritic anterior ischemic optic neuropathy (AION) we investigated the long-term effect of hemodilution on functional results, recurrence rate in the affected and involvement of the second eye. PATIENTS AND METHODS: In a retrospective study we reviewed 24 patients ranging in age from 62 to 92 years (mean 77 years) with AION. The duration of follow-up was between 8-51 months (mean 24.8 months). All patients received iso- (Hct > 40%) or hypervolemic (Hct < or = 40%) hemodilution over 8-10 days with daily infusion of 10% hydroxyethyl-starch 200/0.5. The hematocrit decreased significant (p < 0.001) from 43.4 +/- 3.8% to 37.8 +/- 3.5% after this 10-day hemodilution treatment. RESULTS: After the longterm follow-up 29.1% of the patients had an improvement of the central vision by two or more lines, 16.7% had a deterioration, 54.2% remained unchanged. The resulting visual acuity was between 0.5-1.0 in 37.5%, between 0.4-0.1 in 41.7% and worse than 0.1 in 20.8%. Neither visual fields nor the VECP were significant changed by hemodilution. After the mean follow-up period of 24.8 months no recurrence occurred in the affected eye and the second eye as never involved. CONCLUSION: Hemodilution therapy has no significant longterm effect on visual acuity and visual fields, but it seems to have a beneficial influence on the recurrence rate in the affected and on the involvement of the second eye.

Destruction of human tear proteins by ozone.

By an in vitro system human tears from healthy donors were exposed in a sealed vessel to a single dose of ozone. A marked destruction of tear proteins by ozone was observed as indicated by the disappearance of the protein pattern in polyacrylamide gel electrophoresis compared with the control. Lysozyme, a significant protein in tears, was demonstrated to be susceptible to degradation by O3. The destruction of tear proteins by ozone can be determined quantitatively by radial immunodiffusion as shown for human tear albumin. Variations of ozone concentrations and reaction time showed that destruction of tear proteins by ozone depended on the ozone concentration and occurred within a few minutes of exposure of tears to ozone.

Degradation of hyaluronate by the concerted action of ozone and sunlight.

The influence of ozone and sunlight in a concerted reaction on hyaluronate solutions was investigated. The kinematic viscosity of hyaluronate solutions is decreased by ozone-air mixtures and simultaneous radiation with sun rays within a few minutes, indicating a depolymerization of the hyaluronate molecule. The reaction is dependent on the concentration of ozone and on the time of exposure to ozone and sunlight. The concerted degradation of hyaluronate is more effective than the reaction with each component, ozone and sun rays, alone. We conclude that hyaluronate depolymerization by ozone and sunlight may be one factor for irritations of the eye by photochemical smog and increased exposure to sun rays.

Effect of calcium dobesilate on the inhibition of collagen fibrillogenesis by glucose in vitro.

Glucose inhibits collagen fibril formation as shown in an in vitro system using thermal fibrillogenesis of collagen type I and type II solutions containing different concentrations of glucose. Studies of the time course and extent of fibril formation indicate that glucose values similar to subjects with normal glucose metabolism effect no significant difference to the control without glucose, whereas high glucose levels mimicking glucose concentrations of diabetics with poor glucose control, significantly delay collagen fibrillogenesis. By defined concentrations of calcium dobesilate (Doxium) the effect of a pathologically high glucose content of collagen fibril formation can be inversed to parameters similar as observed with glucose concentrations of a subject with normal glucose metabolism.

Reaggregation behavior of different types of collagen in vitro: variations in the occurrence and structure of dimeric segment long-spacing collagen.

Segment long-spacing collagen (SLS) can be precipitated from solutions of collagen using ATP as the inducing agent. Dimeric SLS aggregates have been observed in addition to monomeric SLS. We have compared collagen types I, II, III, and V with respect to their ability to form dimeric SLS in vitro. These collagen types were isolated from bovine tissues and characterized by polyacrylamide slab gel electrophoresis of the respective alpha-chains. Only monomeric SLS can be detected in preparations of collagen types I and III. Dimeric SLS, on the other hand, accounts for the majority of the crystallites seen in preparations of collagen types II and V. Dimeric SLS from both collagen types II and V reveal overlap zones at the carboxy-terminal ends of the collagen molecules. However, dimeric SLS from collagen types II and V differ with respect to their overlap distances. Significant portions of the triple helical domains of collagen molecules are occupied by the overlap region of dimeric SLS from type II collagen. On the other hand, dimeric SLS from type V collagen is composed of molecules overlapping only at their short nonhelical telopeptides. It is concluded that the ability of collagen molecules to aggregate into dimeric SLS under defined experimental conditions is collagen type dependent.

[PMN elastase determination in tear fluid in corneal ulcer]. / PMN-Elastase-Bestimmung in der Tränenflüssigkeit bei Ulcus corneae.

The PMN-Elastase concentration in tear fluid was determined in 87 persons without eye disease and compared with the PMN-elastase values of 28 patients with ulcus corneae. A significant increase in the PMN-elastase concentration was found in patients with ulcus corneae (means = 1196 micrograms/l) in comparison with the control group (means = 73 micrograms/l). The lapse control shows that the PMN-elastase values in the tear fluid of patients with ulcus corneae are elevated for a number of weeks, even though there are clinical signs of healing of the ulcer. The determination of PMN-elastase in tear fluid represents a new parameter indicating the real end of the inflammatory processes in the eye.