Oxidative stress-induced retinal damage is prevented by mild hypothermia in an ex vivo model of cultivated porcine retinas.

BACKGROUND: Hydrogen peroxide (H2 O2 ) can be used in vitro to simulate oxidative stress. In retinal organ cultures, H2 O2 induces strong neurodegeneration of the retina. It is known that oxidative stress plays a role in the development of several retinal diseases including glaucoma and ischemia. Thus, we investigated whether processes underlying oxidative stress can be prevented by hypothermia using an ex vivo organ culture model of porcine retinas. METHODS: Porcine retinal explants were cultivated for 5 and 8 days. Oxidative stress was induced via 300 µM H2 O2 on day 1 for 3 hours. Hypothermia treatment at 30°C was applied simultaneously with H2 O2 , for 3 hours. Retinal ganglion cells (RGCs), apoptosis, bipolar and cholinergic amacrine cells, microglia and macroglia were evaluated immunohistologically. Apoptosis rate was additionally analysed via western blot. RESULTS: Reduced apoptosis rates through hypothermia led to a preservation of RGCs (P < .001). Amacrine cells were rescued after hypothermia treatment (P = .17), whereas bipolar cells were only protected partly. Additionally, at 8 days, microglial response due to oxidative stress was completely counteracted via hypothermia (P < .001). CONCLUSIONS: H2 O2 induced strong degenerative processes in porcine retinas. The role of oxidative stress in the progression of retinal diseases makes this ex vivo organ culture model suitable to investigate new therapeutic approaches. In the present study, the damaging effect of H2 O2 to several retinal cell types was counteracted or strongly alleviated through hypothermia treatment. Especially RGCs, which are affected in glaucoma disease, were protected due to a reduced apoptosis rate through hypothermia.

Hypothermia protects retinal ganglion cells against hypoxia-induced cell death in a retina organ culture model.

BACKGROUND: Hypoxia contributes to retinal damage in several retinal diseases, including central retinal artery occlusion, with detrimental consequences like painless, monocular loss of vision. Currently, the treatment options are severely limited due to the short therapy window, as the neuronal cells, especially the retinal ganglion cells (RGCs), are irreversibly damaged within the first few hours. Hypothermia might be a possible treatment option or at least might increase the therapy window. METHODS: To investigate the neuroprotective effect of hypothermia after retinal hypoxia, an easy-to-use ex vivo retinal hypoxia organ culture model developed in our laboratory was used that reliably induced retinal damage on a structural, molecular and functional level. The neuroprotective effect of hypothermia after retinal hypoxia was analysed using optical coherence tomography scans, histological stainings, quantitative real-time polymerase chain reaction, western blotting and microelectrode array recordings. RESULTS: Two different hypothermic temperatures (30°C and 20°C) were evaluated, both exhibited strong neuroprotective effects. Most importantly, hypothermia increased RGC survival after retinal hypoxia. Furthermore, hypothermia counteracted the hypoxia-induced RGC death, reduced macroglia activation, attenuated retinal thinning and protected from loss of spontaneous RGC activity. CONCLUSIONS: These results indicate that already a mild reduction in temperature protects the RGCs against damage and could function as a promising therapeutic option for hypoxic diseases.

Establishment of a porcine corneal endothelial organ culture model for research purposes.

Human corneas usually are not available for research, as they are used for transplantation only. At the same time, scientific studies on cultured human endothelial cells can produce misleading results due to inevitable dedifferentiation. Therefore, an organ-culture model of porcine corneas-displaying endothelial cell death rates comparable to those of cultured human corneas-would be very desirable. Fresh pig eyes were prepared under sterile conditions to obtain corneoscleral buttons, corneal buttons and so called "split corneal buttons" (new preparation method) and cultivated for 15 days. Morphology of the endothelial cell layer was observed by light microscopy on day 1, 8 and 15. On day 15 staining with trypan blue and alizarin red S was performed. Photographs were evaluated in a randomized, blinded manner. Here, the morphology of the corneal endothelium and the number of endothelial cells per mm2 were analyzed. After 15 days of cultivation the endothelial cell layer was maintained only in corneal buttons and split corneal buttons. Alizarin red S stained areas and the existence of polymorphisms like rosette figures and reformation figures were significantly less frequent in split corneal buttons than in corneal buttons. Loss of endothelial cells was significantly greater in corneal buttons [575 ± 25/250 cells/mm2 (median ± 25%/75%-quantile); 14.8%] than in split corneal buttons [417 ± 138/179 cells/mm2 (median ± 25%/75%-quantile); 10.2%]. The new preparation method of split corneal buttons allows the cultivation of porcine corneas for 2 weeks with cell death rates comparable to those of the corresponding human tissue in cornea banks without the need to add de-swelling additives to the media. This is therefore a simple and highly reliable method model to be applied in intervention studies on corneal endothelial cells in their natural compound.

Pars plana vitrectomy for treatment of advanced Coats' disease--presentation of a modified surgical technique and long-term follow-up.

BACKGROUND: To present a modified surgical technique in the treatment of retinal detachment secondary to advanced Coats' disease in children, and report on long-term anatomical and functional outcome. METHODS: We analysed an interventional case series of 13 patients (13 eyes) with advanced Coats' disease characterised by retinal detachment in addition to massive subretinal exudates and vascular malformation. The presented patients underwent pars plana vitrectomy (PPV), including a modified technique of exocryotherapy applied after fluid-air exchange in order to achieve complete treatment of the vascular changes, to reduce associated side-effects, and to avoid retinectomy and silicone oil tamponade. RESULTS: Within a median follow-up period of 37 months (range: 18-66 months), no enucleation was necessary. Four eyes (31 %) did not need any further therapy, and in nine eyes (69 %) additional treatments were performed. Six patients (46 %) required revisional surgery with silicone oil tamponade. In ten eyes (77 %), the pathologic vessels and exudates finally regressed and the retina reattached. Visual acuity (VA) could be stabilized in the majority of patients: in three eyes (27 %) VA improved, in four eyes (36 %) VA remained stable, in four eyes (36 %) visual acuity (VA) deteriorated, and in two eyes VA could not be evaluated. CONCLUSIONS: The presented modified technique allows for sufficient cryotherapy of vascular malformations, even in the presence of massive exudation, in a subset of patients with advanced Coats' disease, and thus may reduce surgery-related complications and improve the rehabilitation process of these young patients.

Early postoperative changes of the foveal surface in epiretinal membranes: comparison of 23-gauge macular surgery with air vs. balanced salt solution.

OBJECTIVE: To analyze the foveal surface using binary image analysis after spectral-domain optical coherence tomography (SD-OCT) following 23-gauge macular surgery in epiretinal membranes (ERM) using either air tamponade (AIR) or balanced salt solution (BSS). METHODS: One hundred twenty-four eyes (124 patients) with ERM that had undergone membrane peeling with installation of air or BSS were analyzed retrospectively. Ophthalmic examination was performed at baseline and 3 months. OUTCOME MEASURES: The foveal area and surface symmetry, area matched thickness, area matched contour, and best-corrected visual acuity (BCVA). The OCT images were analyzed after binary conversion with ImageJ software. RESULTS: Eighty eyes (80 patients) of 124 screened patients were included (AIR group: 39 patients, BSS group: 41 patients). Median follow-up time was 14 weeks (range, 9-19 weeks). Three months after surgery, the median horizontal area decreased significantly in both groups (p < 0.0001). At follow-up, the foveal surface symmetry values for the BSS group (median, 22.73 µm, range, 0-153) were significantly lower than for the AIR group (median, 23.95 µm, range, 0-160.43) (p < 0.0001). The area-matched thickness increased significantly in both groups (p < 0.001). The AIR group showed a significant increase of the area matched contour for the nasal located measurement-areas N1 (p < 0.0003), N2 (p < 0.0079), N3 (p < 0.007). The BSS group showed a significant increase of the area-matched contour for the measurement areas N1 (p < 0.019), N2 (p < 0.0014), and N4 (p < 0.022). After surgery, median BCVA for both groups increased significantly to 0.3 logMAR. CONCLUSIONS: The analysis of early contour changes after ERM surgery was technically possible. Long-term data have to be looked at before the clinical impact of this methodology can be estimated. Although there were no big differences between both groups (AIR vs. BSS), this could change within a longer and more representative follow-up.

Critical evaluation of the usability of augmented reality ophthalmoscopy for the training of inexperienced examiners.

PURPOSE: To measure the value of augmented reality technology usage to teach the medical students performing binocular indirect ophthalmoscopy. METHODS: Thirty-seven medical students were randomly assigned to the training of binocular indirect ophthalmoscopy either in the conventional way or with augmented reality ophthalmoscopy (ARO). For testing student's skills, they had to examine a real person using a conventional ophthalmoscopy system and draw the optic disk. They also had to fill out a questionnaire. Subjective and objective evaluations were performed. RESULTS: Thirty-seven students were randomly assigned to two groups. Eighteen students were trained with conventional ophthalmoscopy and 19 students with ARO. The questionnaires showed no differences. Performing an objective analysis, the median ophthalmoscopy training score for the conventional ophthalmoscopy group was 1.2 (range, 0.67-2) and showed a significant difference (P < 0.0033) to the ARO group (median 2; range, 0.67-2). CONCLUSION: The study provides evidence that a single ARO training is efficient to improve ophthalmoscopy skills. As the objective analysis showed, the ARO group had a significantly superior performance. Our study also indicates that subjective evaluation of the fundus drawings without systematic analysis is prone to errors.

DMEK outcome after one year - Results from a large multicenter study in Germany.

PURPOSE: Descemet membrane endothelial keratoplasty (DMEK) accounts for >50% of all corneal transplants in Germany. So far, no data from such a large multicenter study have been published. METHODS: This retrospective study included 3200 DMEKs at seven departments performed for Fuchs endothelial corneal dystrophy (FECD) or bullous keratopathy (BK). We evaluated best corrected visual acuity (BCVA, logMAR), endothelial cell density (ECD, cells/mm2 ), minimal corneal thickness (CT, µm), rebubbling-, primary transplant failure- and immune reaction-rate. Changes over time were evaluated by linear mixed models for repeated measures and correlation with case number by center by weighted linear regression. RESULTS: For patients without vision-limiting comorbidities (74% of all analysed eyes, n = 2270), mean BCVA improved from 0.6 ± 0.4 logMAR to 0.2 ± 0.2 logMAR 6 months (p < 0.001, n = 1441) and 0.1 ± 0.2 logMAR 12 months (p = 0.001, n = 1402) postoperatively. BK- had a worse BCVA compared to FECD-patients (0.3 ± 0.5 vs. 0.1 ± 0.2 logMAR [p < 0.001] at 1 year). ECD declined from 2465 ± 259 cells/mm2 (n = 2876 preoperatively) to 1587 ± 433 cells/mm2 after 12 months (p < 0.001, n = 1237). Mean rebubbling rate was 0.4 ± 0.7/eye. 784 eyes (25%) received at least one rebubbling. More rebubblings correlated with a lower ECD, a worse BCVA, a higher CT, and higher transplant failure and rejection rates (p < 0.001, p = 0.013 for BCVA at 12 months). A single rebubbling did not influence the BCVA (p = 0.785). Graft failure rate was 3% (n = 67), rejection rate 1.5% (n = 48). CONCLUSION: Descemet membrane endothelial keratoplasty increases visual acuity with low transplant failure- and rejection-rates. FECD has a better outcome than BK. Since a quarter of all patients need a rebubbling, this should be included in the informed consent. Remarkably, one rebubbling has no influence on the outcome.

GDF-15: a novel serum marker for metastases in uveal melanoma patients.

BACKGROUND: About 50% of patients with uveal melanoma (UM) develop metastases during the course of their disease. We analyzed serum levels of Growth Differentiation Factor-15 (GDF-15), with the aim of identifying patients with early metastases. METHODS: GDF-15 concentration was measured using an enzyme-linked immunosorbent assay (ELISA) in serum samples from 188 UM patients (170 patients without metastases; 18 patients with clinically detectable metastases) and 18 healthy control individuals. Data were analyzed with respect to differences between patients with and without clinically detectable UM metastases. GDF-15 serum levels were further analyzed with regard to significant patient and tumor characteristics as revealed by histology and multiplex ligation-dependent probe amplification (MLPA) to determine chromosome 3 copy number. GDF-15 expression in UM was investigated by immunohistochemistry. RESULTS: Patients with clinically detectable metastases had significantly higher GDF-15 serum levels compared to those without clinically detectable metastases as well as to healthy individuals (ANOVA; p < 0.001). GDF-15 concentrations in UM patients with overt clinically detectable metastases were significantly higher than those in UM patients with a second malignancy in remission but without clinically detected UM metastases (ANOVA; p < 0.001). No association between serum concentration of GDF-15 and clinical, pathological, and genetic features was observed. GDF-15 protein was only expressed in a minority of UM cells in most tumors. CONCLUSIONS: Our data suggest that GDF-15 can be used as a serum marker for the diagnosis of metastases in UM patients. Further data collection and analysis are necessary to evaluate a possible prognostic role of GDF-15 in predicting early metastases.

Spatial and temporal resolution of the photoreceptors rescue dynamics after treatment with voretigene neparvovec.

BACKGROUND: Voretigene neparvovec is a gene therapeutic agent for treatment of retinal dystrophies caused by bi-allelic RPE65 mutations. In this study, we report on a novel and objective evaluation of a retinotopic photoreceptor rescue. METHODS: Seven eyes of five patients (14, 21, 23, 24, 36 years, 1 male, 4 females) with bi-allelic RPE65 mutations have been treated with voretigene neparvovec. The clinical examinations included visual acuity testing, dark-adapted full-field stimulus threshold (FST), dark-adapted chromatic perimeter (DAC) with a 30-degree grid, and a 30 degrees grid scotopic and photopic chromatic pupil campimetry (CPC). All evaluations and spectral domain optical coherence tomography were performed at baseline, 1 month and 3 months. RESULTS: All except the oldest patient had a measurable improvement of the rod function assessed via FST, DAC or scotopic CPC at 1 month. The visual acuity improved slightly or remained stable in all eyes. A cone function improvement as measured by photopic CPC was observed in three eyes. The gain of the dark-adapted threshold with blue FST and the DAC stimuli (cyan) average correlated strongly with age (R2>0.7). The pupil response improvement in the scotopic CPC correlated with the baseline local retinal volume (R2=0.5). CONCLUSIONS: The presented protocols allow evaluating the individual spatial and temporal effects of gene therapy effects. Additionally, we explored parameters that correlated with the success of the therapy. CPC and DAC present new and fast ways to assess functional changes in retinotopic maps of rod and cone function, measuring complementary aspects of retinal function.

Intravitreal bevacizumab for treatment of serous macular detachment in central retinal vein occlusion.

PURPOSE: To report the long-term effect of intravitreal bevacizumab on serous macular detachment (SMD) in central retinal vein occlusion (CRVO). METHODS: Retrospective, interventional, noncomparative case series. Nineteen consecutive patients (19 eyes) with SMD secondary to CRVO were included. Primary outcomes were the change of the best-corrected visual acuity (BCVA) and the central foveal thickness (CFT) at final visit. Secondary outcome was the resolution of the SMD. RESULTS: The mean patient age was 65.6 years, and the mean follow-up time 21.6 months. Of the 19 eyes, 15 eyes were non-ischemic. The average number of bevacizumab injections was 5.9 from baseline to the final visit. Mean logMAR BCVA improved from 1.20 ± 0.45 (20/317) to [Formula: see text] and mean CFT decreased from 918 ± 280 µm to [Formula: see text] at the final visit. The SMD resolved in 16 of the 19 eyes completely. No local or systemic complication was observed. CONCLUSION: In this retrospective case series, a significant improvement of the vision and resolution of the SMD was found after bevacizumab treatment for CRVO with SMD. Large case series are necessary to evaluate the role of the intravitreal bevacizumab treatment for CRVO associated with SMD.

Effects of bevacizumab on apoptosis, Na+ -K+ -adenosine triphosphatase and zonula occludens 1 expression on cultured corneal endothelial cells.

BACKGROUND: This laboratory study was undertaken to investigate the influence of bevacizumab on apoptosis, Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase) and zonula occludens 1 (ZO-1) expression on cultured human corneal endothelial cells (HCECs). METHODS: Annexin V binding combined with propidium iodide (PI) costaining was used to distinguish viable, early and late apoptotic cells. Immunolocalization of ZO-1 and Na(+)-K(+)-ATPase was performed to analyze intercellular cell integrity after exposure to 5.0 mg/ml bevacizumab for 24 h. RESULTS: No significant induction of apoptosis or necrosis was seen in HCECs after exposure to 5.0 mg/ml bevacizumab (p = 0.689, p = 0.516, respectively). The mean number of annexin-V-FITC- and PI-positive cells did not change significantly. Additionally, no significant changes in expression were detectable, neither for ZO-1 nor for Na(+)-K(+)-ATPase in comparison with the control. For ZO-1, 70.0% of the cells stained intensely, 24.7% stained moderately, and 5.3% stained weakly in the control group. After exposure to 5.0 mg bevacizumab, only minor changes were observable: 68.8% stained intensely, 25.4% moderately and 5.8% weakly (p = 0.524). For Na(+)-K(+)-ATPase, 19.3% of the cells stained intensely, 59.4% moderately, and 21.3% weakly in the control group. After exposure to 5.0 mg bevacizumab, again only minor changes were observable in the expression pattern: 18.2% stained intensely, 60.3% moderately and 21.5% weakly. The changes were not significant compared with the control (p = 0.492). CONCLUSIONS: Bevacizumab, at concentrations used clinically, did not induce apoptosis or necrosis in HCECs in vitro. Additionally, no alteration of ZO-1 or Na(+)/K(+)-ATPase expression was detected after exposure to 5.0 mg/ml bevacizumab for 24 h.

Long-term visual acuity and its predictors after cataract surgery in patients with uveitis.

PURPOSE: To analyze the outcomes of phacoemulsification and posterior intraocular lens (IOL) implantation in patients with uveitis and to determine factors responsible for poor visual outcome. METHODS: The records of 155 patients (180 eyes) with uveitis who had phacoemulsification and IOL implantation between August 2001 and March 2008 were examined retrospectively. Best-corrected visual acuity (BCVA) was recorded at the immediate preoperative visit and at follow-up examinations every 3 months. At each postoperative visit, a complete ophthalmologic examination was performed. The postoperative visual outcomes and complications were analyzed. Univariate regression analysis was done to determine risk factors for poor visual acuity during follow-up. RESULTS: The mean follow-up was 31.4 months (range 3-78 months). An underlying systemic disease was present in 70 (45.2%) patients (82 eyes, 45.6%). The mean preoperative logMAR BCVA was 1.13 +/- 0.62 (95% CI: 0.85-1.02) and increased to 0.42 +/- 0.57 (95% CI: 0.32-0.59) at last medical visit (p < 0.001). A total of 107 eyes (59.4%) had postoperative complications including posterior capsular opacification, newly developed macular edema, recurrence of uveitis, macular epiretinal membrane, and deposits on the IOL surface. Preoperatively observed macular lesions was the factor most strongly associated with poor visual outcome after cataract surgery (odds ratio: 5.43; 95% CI: 3.41-7.34; p < 0.001). Anterior segment pathologies, age at surgery, etiology of uveitis (idiopathic, uveitis associated systemic disease), and gender did not influence visual rehabilitation after surgery (p > 0.05). CONCLUSIONS: The outcomes of phacoemulsification and IOL implantation in patients with uveitis were satisfactory. Patients with observed preoperative macular lesions are at risk for poor visual outcome.

Effects of ultrasound energy on the porcine corneal endothelium - Establishment of a phacoemulsification damage model.

PURPOSE: The purpose of this study was to establish a standardized in vitro phacoemulsification damage model for future investigations of the effects of phacoemulsification, surgical devices, protective ophthalmic viscoelastic devices (OVDs), irrigation solutions and other aspects related to cataract phacoemulsification surgery on the corneal endothelium using porcine eyes. METHODS: Thirty-four porcine eyes were randomly assigned to three groups (phacoemulsification (n = 13), irrigation (n = 9), control (n = 12)). A total of 5 min of ultrasound energy with intermittent irrigation/aspiration was applied in the eyes of the phacoemulsification group. The eyes of the irrigation group received the identical treatment, but without the application of ultrasound energy. The control group was left untreated. All eyes were then prepared to split corneal buttons followed by 15 days of cultivation. Endothelial cell density (ECD) was assessed blinded on day 15. RESULTS: Endothelial cell density declined significantly more until day 15 in the phacoemulsification group (2567 ± 317/267 cells/mm² (median ± 25%/75%-quartiles), -32.5 ± 7.0/6.4%) compared to the irrigation (3450 ± 350/383 cells/mm², -11.8 ± 5.3/2.6%; p < 0.001) and the control group (3650 ± 288/258 cells/mm², -10.2 ± 3.2/4.6%; p < 0.001). CONCLUSION: The phacoemulsification damage model presented in this study is sensitive to phacoemulsification energy and may reliably be used to investigate various factors involved in phacoemulsification with regard to their influence on corneal endothelial cells. This method is able to replace animal experiments or in vitro cell culture experiments that often do not translate well to the in vivo situation in humans.

Intensified monitoring of circadian blood pressure and heart rate before and after intravitreous injection of bevacizumab: preliminary findings of a pilot study.

BACKGROUND: A dose-dependent increase in arterial blood pressure (BP) was seen during bevacizumab treatment given intravenously for metastatic carcinoma. Because low systemic levels can also be expected after the intravitreal administration of bevacizumab, we looked for possible haemodynamic reactions of patients at higher risk of developing cardiovascular events after bevacizumab injection. METHODS: Ambulatory BP was monitored in 14 hypertensive patients receiving 1.25 mg intraocular bevacizumab for either choroidal neovascularization (CNV) or retinal proliferation associated with central retinal vein occlusion (CRVO). Circadian measurement was carried out twice, first at least 24 h prior to injection and second 72 h afterwards. Baseline evaluation before injection was compared with values taken in a matched control group. Taking a small random sample of two patients, serum concentration of bevacizumab and VEGF-A was measured at several time points. RESULTS: High incidence of pathologic BP values was found in the pre-injection measurement, even under anti-hypertensive treatment of the patients with CNV or CRVO. No general increase in BP was seen after the intravitreal injection (P = 0.01), although significantly reduced nocturnal dipping occurred as compared to before the injection (P = 0.006). Individual patients showed a rise in BP load subsequent to injection. A decline in serum VEGF-A was found to correspond to measureable levels of serum bevacizumab (up to 90 ng/ml). CONCLUSIONS: Before the intravitreal injection, BP values were increased in the majority of the patients. The elevated BP load might be related to probable pre-injection stressors. There seems to be no general rise in mean BP, heart rate and pulse pressure after intravitreal bevacizumab, although a decrease in serum VEGF-A can occur in individual patients. The reduced nocturnal dipping could be caused by pharmacodynamic effects on the vasal tone; this preliminary but striking finding warrants further investigation.

A new knotless technique for combined transscleral fixation of artificial iris, posterior chamber intraocular lens, and penetrating keratoplasty.

PURPOSE: To present a new surgical technique for treating corneal opacity and aniridia with aphakia and the results in a small consecutive case series. METHODS: A three-piece acrylic intraocular lens (IOL) was attached to a customized silicone iris prosthesis and fixed with three 10-0 polypropylene sutures in a knotless technique using Z-sutures after trephination of the recipient cornea. The medical records of all consecutive patients who had received a keratoplasty and an implantation of an artificial iris and IOL were reviewed. RESULTS: Five eyes of five patients were included in the analysis. The mean age of the patients was 46.2 years and the mean follow-up was 24.6 months. The mean best-corrected visual acuity improved from 1.36 logMAR before surgery to 0.78 logMAR after surgery during the follow-up. At the last follow-up visit, the artificial iris-IOL complex was well centered with good positioning in all cases. CONCLUSIONS: Management of post-traumatic aniridia combined with aphakia and corneal scars or graft failure by haptic fixation of a foldable IOL on an artificial iris combined with a simultaneous keratoplasty appears to be a promising approach, which allows to correct a complex lesion with a less traumatic and faster procedure.

Diminished apoptosis in hypoxic porcine retina explant cultures through hypothermia.

Simulation of hypoxic processes in vitro can be achieved through cobalt chloride (CoCl2), which induces strong neurodegeneration. Hypoxia plays an important role in the progression of several retinal diseases. Thus, we investigated whether hypoxia can be reduced by hypothermia. Porcine retinal explants were cultivated for four and eight days and hypoxia was mimicked by adding 300 µM CoCl2 from day one to day three. Hypothermia treatment (30 °C) was applied simultaneously. Retinal ganglion, bipolar and amacrine cells, as well as microglia were evaluated via immunohistological and western blot analysis. Furthermore, quantitative real-time PCR was performed to analyze cellular stress and apoptosis. In addition, the expression of specific marker for the previously described cell types were investigated. A reduction of ROS and stress markers HSP70, iNOS, HIF-1α was achieved via hypothermia. In accordance, an inhibition of apoptotic proteins (caspase 3, caspase 8) and the cell cycle arrest gene p21 was found in hypothermia treated retinae. Furthermore, neurons of the inner retina were protected by hypothermia. In this study, we demonstrate that hypothermia lowers hypoxic processes and cellular stress. Additionally, hypothermia inhibits apoptosis and protects neurons. Hence, this seems to be a promising treatment for retinal neurodegeneration.

Toxic effects of recombinant tissue plasminogen activator on cultured human corneal endothelial cells.

PURPOSE: Human corneal endothelial cells (HCECs) are nonmitotic cells. Any intracameral application of a drug requires evaluation of the potential apoptotic and toxic effects. Intracameral recombinant tissue plasminogen activator (rtPA) is successfully used for the treatment of severe and prolonged postoperative fibrin reaction. This study was undertaken to investigate the toxicity of rtPA on cultured HCECs to determine its safety for clinical use. METHODS: Cell cultures of HCECs were harvested from human donor eyes and exposed to various concentrations of rtPA (10-200 microg/mL). For cytotoxicity testing, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and the live/dead viability/cytotoxicity assay were performed. Annexin V binding combined with propidium iodide (PI) costaining was used for the distinction of viable, early, and late apoptotic cells. Odds ratios (ORs) and confidence intervals (CIs) were calculated for 50 microg/mL, 100 microg/mL, and 200 microg/mL. Cell morphology was studied after 24 hours of exposure to rtPA to identify cellular damage. Immunolocalization of zonula occludens 1 (ZO1) was performed to analyze intercellular barrier disturbance in the presence of rtPA. RESULTS: Reduction of mitochondrial dehydrogenase activity after rtPA exposure was dose dependent and suggested comparable toxicity with the data obtained from the live/dead assay. The mean number of Annexin V-FITC and PI-positive cells was not significantly increased at concentrations of 50 microg/mL and 100 microg/mL. At 200 microg/mL, however, the ORs were 5.082 +/- 0.213 (95% CI, 3.962-6.203; P < 0.001) for apoptosis and 6.154 +/- 0.196 (95% CI, 5.123-7.181; P < 0.001) for necrosis. In addition, increasing concentrations of rtPA resulted in a fading immunopositive staining for ZO1. CONCLUSIONS: These data suggest a dose-dependent toxic effect of rtPA on HCECs in vitro. Although intracameral rtPA concentrations up to 100 mug/mL seem to be clinically safe, the use of concentrations higher than 125 microg/mL might induce irreversible cell death and should be restricted to selected cases.

Early effects of triamcinolone on vascular endothelial growth factor and endostatin in human choroidal neovascularization.

OBJECTIVE: To evaluate the early effects of triamcinolone acetonide as monotherapy or as an adjuvant to ocular verteporfin photodynamic therapy (PDT) on angiogenesis in human choroidal neovascularization (CNV) secondary to age-related macular degeneration. METHODS: Retrospective review of an interventional series of 55 patients who underwent CNV extraction. Eleven patients were treated with intravitreal triamcinolone acetonide (4 mg) monotherapy (triamcinolone-treated CNV group [n = 5]) or with PDT-triamcinolone combination therapy (PDT-triamcinolone-treated CNV group [n = 6]) 3 to 9 days before surgery. Forty patients who underwent CNV extraction without previous therapy (control CNV group) and 4 patients who underwent CNV extraction 3 days after PDT (PDT CNV group) served as control subjects. The CNV samples were stained for CD34, endostatin, cytokeratin 18, and vascular endothelial growth factor (VEGF). RESULTS: Vascular endothelial growth factor expression was stronger in the PDT CNV samples (P < .001), triamcinolone CNV samples (P = .01), and PDT-triamcinolone CNV samples (P = .007) compared with the control CNV samples. There were no statistically significant differences in VEGF expression among the PDT CNV samples, triamcinolone CNV samples, and PDT-triamcinolone CNV samples. Endostatin expression was weaker in the PDT CNV samples than in the control CNV samples (P = .008). Endostatin expression was stronger in the triamcinolone CNV samples and the PDT-triamcinolone CNV samples compared with the control CNV samples (P = .001 and P < .001, respectively) and the PDT CNV samples (P < .001 for both). CONCLUSION: To some extent, triamcinolone monotherapy seems to exert its angiogenesis inhibitory effects on CNV by enhancing endostatin expression rather than by suppressing VEGF expression.

Comparison of in vitro safety profiles of vancomycin and cefuroxime on human corneal endothelial cells for intracameral use.

PURPOSE: To evaluate and compare the cytotoxic and apoptotic properties of cefuroxime and vancomycin on cultured human corneal endothelial cells (HCECs) to determine their safety for intracameral use. METHODS: Human corneal endothelial cells were harvested from human donor eyes and exposed to various concentrations of cefuroxime and vancomycin (0.15 to 15 mg/mL). For cytotoxicity testing, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was performed. Annexin V binding combined with propidium iodide (PI) co-staining was used for the distinction of viable, early, and late apoptotic cells. Odds ratios (ORs) and confidence intervals were calculated for the control group (without drug exposure) for 2.75 mg/mL and 15 mg/mL. Cell morphology and immunolocalization of zonula occludens 1 (ZO1) were assessed after 24 hours of drug exposure. RESULTS: Reduction in cell viability was observed in a dose-dependent manner after exposure to both drugs. Cefuroxime concentrations higher than 2.75 mg/mL and vancomycin concentrations higher than 5.0 mg/mL led to significant reduction in cell viability. The mean number of annexin V-positive and PI-positive cells was not significantly increased at 2.75 mg/mL for either antibiotic agent. After exposure to 15.0 mg/mL, however, the late apoptotic/necrotic cells predominated, with higher ORs indicating accelerated cell death. Increasing concentrations of both antibiotic agents resulted in fading immunopositivity for ZO1. CONCLUSIONS: These data suggest a dose-dependent toxicity of cefuroxime and vancomycin on HCECs in vitro with a narrow range of safety. Although the clinically used concentrations seem to be safe, slightly higher concentrations might induce irreversible cell death and thus should be avoided.

Ocular surface squamous neoplasia as the first apparent manifestation of HIV infection in Malawi.

PURPOSE: To evaluate the prevalence of undiagnosed and asymptomatic HIV infection in patients with ocular surface squamous neoplasia (OSSN) in an urban patient population in Malawi. METHODS: A consecutive series of patients presenting with OSSN was evaluated in an African academic centre. A detailed history and physical examination in 53 consecutive patients with conjunctival squamous cell carcinoma and conjunctival intraepithelial neoplasia were performed. Thirty eight (72%) patients agreed to undergo serological HIV testing. RESULTS: Seventy-nine per cent (30 of 38) patients were HIV positive. None of the patients had previous HIV testing or was aware of having symptoms of HIV. Seventy per cent (n = 21) of the HIV-positive patients had no other symptoms suggestive of HIV infection or any other disease. Patients were far more likely to refuse HIV testing if they were married and male. CONCLUSIONS: The conjunctival tumour may be the primary and only apparent manifestation of HIV in patients presenting with OSSN in Sub-Saharan Africa.