The pro-inflammatory role of high-mobility group box 1 protein (HMGB-1) in photoreceptors and retinal explants exposed to elevated pressure.

To determine the role of high-mobility group box 1 protein (HMGB-1) in cellular and tissue models of elevated pressure-induced neurodegeneration, regeneration, and inflammation. Mouse retinal photoreceptor-derived cells (661W) and retinal explants were incubated either under elevated pressure or in the presence of recombinant HMGB-1 (rHMGB-1) to investigate the mechanisms of response of photoreceptors. Immunohistochemistry, western blotting, and the quantitative real-time PCR were used to examine the expression levels of immunological factors (eg, HMGB-1, receptor for advanced glycation end products (RAGE)), Toll-like receptors 2 and 4 (TLR-2, TLR-4), apoptosis-related factors (eg, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated death promoter (Bad)) as well as cytokine expression (eg, tumor necrosis factor alpha (TNF-α), interleukin (IL)-4, IL-6, and vascular endothelial growth factor (VEGF)). The data revealed increased the expression of HMGB-1 and its receptors RAGE, TLR-2, and TLR-4, and TNF-α as well as pro-apoptotic factors (eg, Bad) as well as apoptosis in 661W cells exposed to elevated pressure. Co-cultivation of 661W cells with rHMGB-1 increased the expression levels of pro-apoptotic Bad and cleaved Caspase-3 resulting in apoptosis. Cytokine array studies revealed an increased release of TNF-α, IL-4, IL-6, and VEGF after incubation of 661W cells with rHMGB-1. Upregulation of HMGB-1, TLR-2, and RAGE as well as anti-apoptotic Bcl-2 expression levels was found in the retinal explants exposed to rHMGB-1 or elevated pressure. The results suggest that HMGB-1 promotes an inflammatory response and mediates apoptosis in the pathology of photoreceptors and retinal homeostasis. HMGB-1 may have a key role in ongoing damage of retinal cells under conditions of elevated intraocular pressure.

Optical coherence tomography, scanning laser polarimetry and confocal scanning laser ophthalmoscopy in retinal nerve fiber layer measurements of glaucoma patients.

PURPOSE: To determine the correlations and strength of association between different imaging systems in analyzing the retinal nerve fiber layer (RNFL) of glaucoma patients: optical coherence tomography (OCT), scanning laser polarimetry (SLP) and confocal scanning laser ophthalmoscopy (CSLO). MATERIALS AND METHODOLOGY: 114 eyes of patients with moderate open angle glaucoma underwent spectral domain OCT (Topcon SD-OCT 2000 and Zeiss Cirrus HD-OCT), SLP (GDx VCC and GDx Pro) and CSLO (Heidelberg Retina Tomograph, HRT 3). Correlation coefficients were calculated between the structural parameters yielded by these examinations. The quantitative relationship between the measured RNFL thickness globally and for the four regions (superior, inferior, nasal, temporal) were evaluated with different regression models for all used imaging systems. RESULTS: The strongest correlation of RNFL measurements was found between devices using the same technology like GDx VCC and GDx Pro as well as Topcon OCT and Cirrus OCT. In glaucoma patients, the strongest associations (R²) were found between RNFL measurements of the two optical coherence tomography devices Topcon OCT and Cirrus OCT (R² = 0.513) and between GDx VCC and GDx Pro (R² = 0.451). The results of the OCTs and GDX Pro also had a strong quantitative relationship (Topcon OCT R² = 0.339 and Cirrus OCT R² = 0.347). GDx VCC and the OCTs showed a mild to moderate association (Topcon OCT R² = 0.207 and Cirrus OCT R² = 0.258). The confocal scanning laser ophthalmoscopy (HRT 3) had the lowest association to all other devices (Topcon OCT R² = 0.254, Cirrus OCT R² = 0.158, GDx Pro R² = 0.086 and GDx VCC R² = 0.1). CONCLUSION: The measurements of the RNFL in glaucoma patients reveal a high correlation of OCT and GDx devices because OCTs can measure all major retinal layers and SLP can detect nerve fibers allowing a comparison between the results of this devices. However, CSLO by means of HRT topography can only measure height values of the retinal surface but it cannot distinguish between different retinal layers. This may explain the rather poor correlations and associations between CSLO measurements and those of all other imaging devices which makes it difficult to compare HRT 3 nerve fiber data. These correlations are important in clinical routine especially when different techniques are used in the follow-up of glaucoma patients.

Trabeculectomy Improves Vessel Response Measured by Dynamic Vessel Analysis (DVA) in Glaucoma Patients.

PURPOSE: To determine the effects of surgical IOP reduction (trabeculectomy) on retinal blood flow parameters in glaucoma patients using Dynamic Vessel Analysis (DVA). METHODS: 26 eyes of 26 patients with progressive primary open-angle glaucoma (POAG) despite maximal topical therapy were examined before and after trabeculectomy. The responses of the retinal vessels to flickering light provocation were measured with DVA the day before surgery and 4 to 6 weeks after trabeculectomy. Between 3 and 4 weeks before surgery all local therapies were stopped and a systemic therapy with acetazolamide and conservative free topic steroidal eye drops was started. RESULTS: In 19 patients (73%), an inadequate response to the flicker stimulation was measured preoperatively. In these patients, the maximum dilation of arteries and veins was reduced significantly as compared to healthy eyes. In this group, the maximum dilation of the arteries following the flicker provocation improved from 1.4% before to 3.8% following trabeculectomy (p<0.01). In retinal veins, this parameter increased from 3.1% to 4.6% (p<0.05). In the 7 patients whose arterial and venous reactions to flickering light provocation preoperatively did not differ from healthy eyes, there was no significant change after surgery. The initial baseline values of arteries and veins (MU) did not deviate significantly in both groups. CONCLUSION: POAG patients with progressive disease and impaired vascular regulation profit from IOP lowering trabeculectomy concerning vascular reactivity and dilative reserve, indicating a possible improvement of retinal perfusion following effective IOP control. Future studies with long-term follow-up must determine the clinical importance of these findings for the treatment of glaucoma patients.

Laser cyclophotocoagulation enhances the regulative capacity of retinal vessels in glaucoma.

PURPOSE: To determine the effects of laser surgical IOP reduction by means of transscleral cyclophotocoagulation (CPC) on retinal blood flow parameters in glaucoma patients using Dynamic Vessel Analysis (DVA). MATERIALS AND METHODOLOGY: 26 patients (average age: 70 years) with a long history of primary open angle glaucoma underwent CPC. The effect on the reactive capacity of retinal vessels was assessed before and 6-8 weeks after CPC by means of the Dynamic Vessel Analyzer (DVA) using flicker light provocation. RESULTS: In our group of POAG patients, IOP was significantly reduced about approximately 20% by CPC while systemic blood pressure and heart rate were not changed. The most obvious differences between the pre- and postoperative DVA measurements could be observed in the maximal dilation of the retinal arteries which increased from 0.75 % (+/- 0.6) to 3.17 % (+/- 0.5) with an average increase of 2.4 % (p<0.01). In addition, the ability of the arteries for constriction improved significantly (p<0.05) while the dynamic responses of the veins and the initial baseline values (MU) of the vessel diameters did not change. CONCLUSIONS: Our results of DVA measurements after an IOP-lowering laser surgical intervention (CPC) reveal a significant recovery of the regulative capacity of retinal arteries in glaucoma patients that has up to now neither been properly documented nor appreciated. Future studies with long-term follow-up must determine the clinical importance of these findings for the treatment of glaucoma patients.

Aphakia correction with retropupillary fixated iris-claw lens (Artisan) - long-term results.

PURPOSE: To evaluate the technique, safety, and efficacy of the retropupillary implantation of iris-claw intraocular lenses in a long-term follow-up study. PATIENTS AND METHODS: This retrospective study included 31 eyes of 31 patients who underwent an Artisan aphakic intraocular lens implantation between January 2006 and February 2011 at the University Hospital Essen, Essen, Germany and at the Zentrum für Augenheilkunde PD Dr Laube, Düsseldorf, Germany. Preoperative data collected included demographics, etiology of aphakia, previous surgeries, preoperative eye pathology, intraocular pressure, clinical signs of endothelial cell loss, and best corrected visual acuity. Operative data and postoperative outcomes included the best corrected visual acuity, lens position, intraocular pressure, pigment dispersion, clinical signs of endothelial cell loss, development of macular edema, and other complications. RESULTS: Thirty-one patients were included. The mean follow-up was 25.2 months (range: 4-48 months). The mean best corrected visual acuity postoperatively was 0.64 logarithm of the minimum angle of resolution (logMAR) and varied from 0 logMAR to 3 logMAR. Some patients had a low visual acuity preoperatively because of preoperative eye pathologies. In 22 patients the visual acuity improved, in two patients the visual acuity remained unchanged, and seven patients showed a decreased visual acuity. Complications were peaked pupils (n=10) and retinal detachment in one case. Four patients showed an iris atrophy and high intraocular pressure was observed only in one patient. Subluxation of the intraocular lens, endothelial cell loss, and macular edema were not observed. CONCLUSION: The presented long-term results demonstrate that retropupillary iris-claw lens implantation is a safe and effective method for the correction of aphakia in patients without capsule support. This surgical procedure has the advantages of a posterior chamber implantation with a low intraoperative and postoperative risk profile.

Amniotic membrane transplantation ineffective as additional therapy in patients with aggressive Mooren's ulcer.

BACKGROUND: Mooren's ulcer is a severe ulcerative inflammation of the cornea. The exact pathogenesis remains unclear. Therefore many therapies of Mooren's ulcer are recommended in literature. To shed more light on the ongoing question of optimal treatment of severe progressive Mooren's ulcer, we here report on a retrospective case series of patients treated with systemic immunosuppressive therapy and additional amniotic membrane transplantation. METHODS: Medical records from seven patients (eleven eyes), 4 male and 3 female, with severe progressive Mooren's ulcer were analysed retrospectively. The mean follow up was 88.4 ± 80.8 months (range 12-232 month). A HLA-typing was performed in all patients. A systemic immunosuppressive therapy was administered in all patients. The amniotic membrane was transplanted after the base of the ulcer was resected. RESULTS: Multiple amniotic membrane transplantations were necessary in six patients. The visual outcome of all patients was poor. No patient achieved a visual acuity better than 20/630 Snellen chart. Five patients were positive for HLA-DQ2 and four patients were positive for HLA-DR17(3). CONCLUSIONS: The aggressive and highly inflammatory form of Mooren's ulcer is difficult to treat and the progression of the disease is hard to influence positively even under systemic immunosuppressive therapy. Therefore, the main intention of therapy is to achieve a stable epithelialized corneal surface without the risk of perforation. Amniotic membrane transplantation is not able to cure severe forms of Mooren's ulcer. However it supports the immunosuppressive therapy in acute situations as in critical corneal thinning.

Crystallins are regulated biomarkers for monitoring topical therapy of glaucomatous optic neuropathy.

Optic nerve atrophy caused by abnormal intraocular pressure (IOP) remains the most common cause of irreversible loss of vision worldwide. The aim of this study was to determine whether topically applied IOP-lowering eye drugs affect retinal ganglion cells (RGCs) and retinal metabolism in a rat model of optic neuropathy. IOP was elevated through cauterization of episcleral veins, and then lowered either by the daily topical application of timolol, timolol/travoprost, timolol/dorzolamide, or timolol/brimonidine, or surgically with sectorial iridectomy. RGCs were retrogradely labeled 4 days prior to enucleation, and counted. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization mass spectrometry, Western blotting, and immunohistochemistry allowed the identification of IOP-dependent proteomic changes. Genomic changes were scrutinized using microarrays and qRT-PCR. The significant increase in IOP induced by episcleral vein cauterization that persisted until 8 weeks of follow-up in control animals (p<0.05) was effectively lowered by the eye drops (p<0.05). As anticipated, the number of RGCs decreased significantly following 8 weeks of elevated IOP (p<0.05), while treatment with combination compounds markedly improved RGC survival (p<0.05). 2D-PAGE and Western blot analyses revealed an IOP-dependent expression of crystallin cry-ßb2. Microarray and qRT-PCR analyses verified the results at the mRNA level. IHC demonstrated that crystallins were expressed mainly in the ganglion cell layer. The data suggest that IOP and either topically applied antiglaucomatous drugs influence crystallin expression within the retina. Neuronal crystallins are thus suitable biomarkers for monitoring the progression of neuropathy and evaluating any neuroprotective effects.

Clinical transplantation of individualized recipient serum-adapted cornea reduces the risk of graft rejection after keratoplasty.

Corneal diseases cause severe visual impairment that necessitates corneal transplantation and frequently repetitive procedures due to graft rejection. We tested the hypothesis that exposure of donor corneas to recipient serum-derived factors during eye banking triggers a preoperative adaptation that is beneficial for postoperative tolerance. Donor corneas were incubated in a medium containing human serum (HS) obtained in each case from the prospective graft recipient in order to individually expose the donor cornea to the recipient's serum. All recipient serum-adapted corneas (RSACs) fulfilled the clinical criteria required by the national law and were transplanted successfully. The postoperative ophthalmological examination extended up to 8 years. All RSACs were tolerated by their recipients and did not cause postoperative complications and no rejection. Proteomic analysis of corneas cultivated in culture medium containing either fetal calf serum (FCS) that is routinely used for cornea banking or HS revealed different patterns of proteins. HS-cultured corneas showed a greater proteomic similarity with native human corneas than did the FCS-cultured corneas, indicating a differential nutrification of the cultured corneal tissue by HS-derived factors. The clinical results show for the first time that postoperative complications such as tissue intolerance and graft rejection might be managed if the corneal tissue is individually adapted to the recipient's serum trophic factors. This new donor tissue treatment procedure offers incontrovertible advantages and could be adapted for low-risk eyes as well as other transplantable tissues.

Comparison of Spectralis-OCT, GDxVCC and GDxECC in assessing retinal nerve fiber layer (RNFL) in glaucomatous patients.

BACKGROUND: Glaucomatous optic neuropathy is characterized by a progressive loss of retinal ganglion cells (RGCs). The defects in the peripapillary retinal nerve fiber layer (RNFL) have been reported to be the earliest sign of glaucoma. We determined the agreement between RNFL thickness assessments from spectral-domain OCT (Spectarlis HRA + OCT; Heidelberg Engeneering, Heidelberg, Germany), scanning laser polarimetry (SLP) with variable cornea compensation (GDxVCC; Carl Zeiss Meditec, Dublin, CA, USA), and SLP with enhanced cornea compensation (GDxECC; Carl Zeiss Meditec, Dublin, CA, USA) in glaucomatous patients. Furthermore, we investigate the influence of typical scan score (TSS) on the results of GDx assessments. METHODS: The enrolled subjects were devided into different groups by modified HODAPP visual field criteria. The peripapillary RNFL thickness was assessed with the three devices . ANOVA test, Pearson and Spearman correlation coefficient, and Bland-Altman plots were used to analyse the RNFL thickness assessments. RESULTS: Ninety-two eyes from 92 glaucomatous subjects were analysed. These were divided into four groups: preperimetric glaucoma (n = 26), mild glaucoma (n = 18), moderate glaucoma (n = 21), and severe glaucoma (n = 27). For Spectralis-OCT, the average RNFL thickness (mean ± SD) was 99.25 ± 26.31 µm, 80.52 ± 16.63 µm, 71.59 ± 21.15 µm, and 63.85 ± 20.86 µm for preperimetric, mild, moderate, and severe glaucoma respectively. For GDxVCC, the corresponding assessments were 52.63 ± 8.18 µm, 52.95 ± 10.20 µm, 46.77 ± 10.62 µm, and 49.70 ± 13.34 µm. For GDxECC, the assessments were 49.35 ± 6.52 µm, 45.92 ± 7.21 µm, 42.19 ± 8.00 µm, and 39.53 ± 8.45 µm. All Spectralis-GDxVCC and Spectralis-GDxECC differences were statistically significant by ANOVA test. The differences between GDxVCC and GDxECC were statistically significant only for severe glaucoma. There was a highly significant correlation between Spectralis-OCT and GDxECC, as well as Spectralis-OCT and GDxVCC, in assessing the RNFL thickness. The best instrument agreement was found between GDxECC and Spectralis-OCT. The RNFL thickness assessed with Spectralis-OCT and GDxECC showed a better correlation to visual field defects than GDxVCC. Evaluating GDx assessments with typical retardation pattern GDxVCC and GDxECC showed very similar RNFL thickness results. CONCLUSIONS: RNFL thickness assessments between GDxVCC, GDxECC, and Spectralis-OCT cannot be directly compared. The assessments are generally higher with Spectralis-OCT than with GDxVCC and GDxECC, because of differences in method of the devices. The atypical retardation pattern has a major impact on the RNFL thickness results of GDx devices. This must be taken into account when evaluating the assessed RNFL thickness results.

Regulation of retinal proteome by topical antiglaucomatous eye drops in an inherited glaucoma rat model.

Examination of the response of the retinal proteome to elevated intraocular pressure (IOP) and to the pharmacological normalization of IOP is crucial, in order to develop drugs with neuroptorective potential. We used a hereditary rat model of ocular hypertension to lower IOP with travaprost and dorzolamide applied topically on the eye surface, and examine changes of the retinal proteome. Our data demonstrate that elevated IOP causes alterations in the retinal protein profile, in particular in high-mobility-group-protein B1 (HMGB1), calmodulin, heat-shock-protein (HSP) 70 and carbonic anhydrase II expression. The changes of the retinal proteome by dorzolamide or travoprost are different and independent of the IOP lowering effect. This fact suggests that the eye drops exert a direct IOP-independent effect on retinal metabolism. Further investigations are required to elucidate the potential neuroprotective mechanisms signaled through changes of HMGB1, calmodulin, HSP70 and carbonic anhydrase II expression in glaucoma. The data may facilitate development of eye drops that exert neuroprotection through direct pharmacological effect.

Traumatology of the optic nerve and contribution of crystallins to axonal regeneration.

Within a few decades, the repair of long neuronal pathways such as spinal cord tracts, the optic nerve or intracerebral tracts has gone from being strongly contested to being recognized as a potential clinical challenge. Cut axonal stumps within the optic nerve were originally thought to retract and become irreversibly necrotic within the injury zone. Optic nerve astrocytes were assumed to form a gliotic scar and remodelling of the extracellular matrix to result in a forbidden environment for re-growth of axons. Retrograde signals to the ganglion cell bodies were considered to prevent anabolism, thus also initiating apoptotic death and gliotic repair within the retina. However, increasing evidence suggests the reversibility of these regressive processes, as shown by the analysis of molecular events at the site of injury and within ganglion cells. We review optic nerve repair from the perspective of the proximal axon stump being a major player in determining the successful formation of a growth cone. The axonal stump and consequently the prospective growth cone, communicates with astrocytes, microglial cells and the extracellular matrix via a panoply of molecular tools. We initially highlight these aspects on the basis of recent data from numerous laboratories. Then, we examine the mechanisms by which an injury-induced growth cone can sense its surroundings within the area distal to the injury. Based on requirements for successful axonal elongation within the optic nerve, we explore the models employed to instigate successful growth cone formation by ganglion cell stimulation and optic nerve remodelling, which in turn accelerate growth. Ultimately, with regard to the proteomics of regenerating retinal tissue, we discuss the discovery of isoforms of crystallins, with crystallin beta-b2 (crybb2) being clearly upregulated in the regenerating retina. Crystallins are produced and used to promote the elongation of growth cones. In vivo and in vitro, crystallins beta and gamma additionally promote the growth of axons by enhancing the production of ciliary neurotrophic factor (CNTF), indicating that they also act on astrocytes to promote axonal regrowth synergistically. These are the first data showing that axonal regeneration is related to crybb2 movement within neurons and to additional stimulation of CNTF. We demonstrate that neuronal crystallins constitute a novel class of neurite-promoting factors that probably operate through an autocrine and paracrine mechanism and that they can be used in neurodegenerative diseases. Thus, the post-injury fate of neurons cannot be seen merely as inevitable but, instead, must be regarded as a challenge to shape conditions for initiating growth cone formation to repair the damaged optic nerve.

Scanning laser topography and scanning laser polarimetry: comparing both imaging methods at same distances from the optic nerve head.

PURPOSE: To compare the performance of scanning laser topography (SLT) and scanning laser polarimetry (SLP) on the rim of the optic nerve head and its surrounding area and thereby to evaluate whether these imaging technologies are influenced by other factors beyond the thickness of the retinal nerve fiber layer (RNFL). MATERIALS AND METHODOLOGY: A total of 154 eyes from 5 different groups were examined: young healthy subjects (YNorm), old healthy subjects (ONorm), patients with normal tension glaucoma (NTG), patients with open-angle glaucoma and early glaucomatous damage (OAGE) and patients with open-angle glaucoma and advanced glaucomatous damage (OAGA). SLT and SLP measurements were taken. Four concentric circles were superimposed on each of the images: the first one measuring at the rim of the optic nerve head (1.0 ONHD), the next measuring at 1.25 optic nerve head diameters (ONHD), at 1.5 ONHD and at 1.75 ONHD. The aligned images were analyzed using GDx/NFA software. RESULTS: Both methods showed peaks of RNFL thickness in the superior and inferior segments of the ONH. The maximum thickness, registered by the SLT device was at the ONH rim where the SLP device tended to measure the lowest values. SLT measurements at the ONH were influenced by other tissues besides the RNFL like blood vessels and glial tissues. SLT and SLP were most strongly correlated at distances of 1.25 and 1.5 ONHD. CONCLUSIONS: While both imaging technologies are valuable tools in detecting glaucoma, measurements at the ONH rim should be interpreted critically since both methods might provide misleading results. For the assessment of the retinal nerve fiber layer we would like to recommend for both imaging technologies, SLT and SLP, measurements in 1.25 and 1.5 ONHD distance of the rim of the optic nerve head.

GM-CSF protects rat photoreceptors from death by activating the SRC-dependent signalling and elevating anti-apoptotic factors and neurotrophins.

BACKGROUND: The term retinitis pigmentosa (RP) comprises a heterogeneous group of hereditary and sporadic human retinal degenerative diseases. The molecular and cellular events still remain obscure, thus hiding effective therapies. Granulocyte­macrophage colony-stimulating factor (GM-CSF) is a hematopoietic factor which plays a crucial role in protecting neuronal cells. Binding of GM-CSF to its receptor induces several intracellular signaling pathways and kinases. Here we examined whether GM-CSF has a neuroprotective effect on photoreceptor degeneration in Royal College of Surgeons (RCS) rats. METHODS: GM-CSF was injected into the vitreous body of RCS rats either once at the onset of photoreceptor degeneration at day 21, or twice at day 21 and day 42. At day 84, when photoreceptor degeneration is completed, the rats were sacrificed, their eyes enucleated and processed for histological staining and counting the surviving photoreceptor nuclei. The expression of apoptosis-related factors, such as BAD, APAF1 and BCL-2 was examined by Western blot analysis. The expression of neurotrophins such as ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glia-derived neurotrophic actor (GDNF), as well as glial fibrillary acidic protein (GFAP) was analysed by Western blots and immunohistochemistry. The expression of JAK/STAT, ERK1/2 and SRC pathway proteins was assessed by Western blot analysis. RESULTS: GM-CSF protects significantly against photoreceptor degeneration in comparison to control group. After a single injection of GM-CSF at P21, a 4-fold increase of photoreceptors was observed, whereas eyes which received a repeated injection of GM-CSF at P42 showed a 10-fold increase of photoreceptors. Western blot analysis revealed a decreased BAD and an increased pBAD and BCL-2 expression, indicating changed expression profiles of apoptosis-related proteins. Neurotrophic factors examined are up-regulated, whereas GFAP was also modulated. At cell signalling levels, GM-CSF activates SRC-dependent STAT3 which is independent of JAK2, while proteins of the ERK1/2 pathway are not affected. CONCLUSIONS: The data suggest that GM-CSF is a potent therapeutic agent in photoreceptor degeneration caused by mutation of the receptor tyrosine kinase gene (Mertk), and may be also effective in other photoreceptor degeneration.

Comparison of the Colvard, Procyon, and Neuroptics pupillometers for measuring pupil diameter under low ambient illumination.

PURPOSE: To compare three different pupillometers (Colvard, Procyon, and Neuroptics) for determining pupil diameter at 0.04 and 0.4 lux ambient illumination. METHODS: In 92 eyes of 46 healthy volunteers, pupil diameter was measured at 0.04 and 0.4 lux. After dark adaptation for 2 minutes, measurements were performed with each device by two examiners. Interobserver agreement, instrument agreement, and repeatability were analyzed. RESULTS: Mean pupil diameter was 6.63+/-0.68 mm, 6.24+/-1.01 mm, and 6.99+/-0.67 mm at 0.04 lux and 6.22+/-0.74, 4.64+/-1.04, and 6.73+/-0.72 mm at 0.4 lux with the Colvard, Procyon, and Neuroptics pupillometers, respectively. The interobserver disagreement ranged within narrower limits for the Colvard (0.04 lux: -1.0 to 0.5 mm; 0.4 lux: -0.75 to 1.0 mm) and Neuroptics (0.04 lux: -1.0 to 0.5 mm; 0.4 lux: -1.7 to 0.7 mm) than for the Procyon (0.04 lux: -0.74 to 1.14 mm; 0.4 lux -1.82 to 2.4 mm) under both light conditions. Instrument agreement ranged within narrower limits for the Colvard versus Neuroptics (0.04 lux: -1.3 to 0.75 mm; 0.4 lux: -1.55 to 1.40 mm) than for the Neuroptics versus Procyon (0.04 lux: -1.06 to 2.69 mm; 0.4 lux: 0.18 to 3.69 mm) or Colvard versus Procyon (0.04 lux: -0.63 to 2.60 mm; 0.4 lux: -0.32 to 3.13 mm) at both light levels. At 0.04 lux, repeatability showed no measurement difference outside +/-0.5 mm for the Colvard and Neuroptics; for the Procyon, 25% of consecutive measurements showed a difference >+/-0.5 mm. At 0.4 lux, 2.5% of consecutive measurements for the Colvard and 5% for the Neuroptics differed by >+/-0.5 mm; for the Procyon, 13% of measurements differed by more than this amount. CONCLUSIONS: Pupil diameters under both light conditions were largest with the Neuroptics pupillometer and smallest with the Procyon. The most "examiner independent" Procyon pupillometer performed poorly. The underestimation of the pupil diameter might have severe consequences for refractive surgery patients. The Neuroptics pupillometer showed a high interobserver agreement and repeatability and therefore high safety.

GM-CSF regulates the ERK1/2 pathways and protects injured retinal ganglion cells from induced death.

Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) is a potent hematopoietic cytokine. In the present study, we examined whether GM-CSF is neuroprotective in retinal ganglion cells (RGCs). First, we studied the expression of GM-CSF and the GM-CSF-alpha-receptor in rat and human retina and in RGC-5 cells. Then, RGC-5 cells were incubated with apoptosis-inducing agents (e.g., staurosporine, glutamate and NOR3). The cell death was assessed by Live-Death-Assays and apoptosis-related-proteins were examined by immunoblotting. In addition, the expression of phosphorylated ERK1/2-pathway-proteins after incubation with GM-CSF and after inhibiting MEK1/2 with U0126 was analyzed. To assess the in vivo-effect, first staurosporine or GM-CSF plus staurosporine was injected into the vitreous body of Sprague-Dawley rats. In a second axotomy model the optic nerve was cut and GM-CSF was injected into the vitreous body. In both models, the RGCs were labeled retrogradely with either Fluoro-Gold or 4-Di-10-Asp and counted. As a first result, we identified GM-CSF and the GM-CSF-alpha-receptor in rat and human retina as well as in RGC-5 cells. Then, in the RGC-5 cells GM-CSF counteracts induced cell death in a dose-and time-dependent manner. With respect to apoptosis, Western blot analysis revealed a decreased Bad-expression and an increased Bcl-2-expression after co-incubation with GM-CSF. Concerning signaling pathways, incubation with GM-CSF activates the ERK1/2 pathway, whereas inhibition of MEK1/2 with U0126 strongly decreased the phosphorylation downstream in the ERK1/2 pathway, and the antiapoptotic activity of GM-CSF in vitro. Like in vitro, GM-CSF counteracts the staurosporine-induced cell death in vivo and protects RGCs from axotomy-induced degeneration. Our data suggest that GM-CSF might be a novel therapeutic agent in neuropathic disease of the eye.

A coculture assay to visualize and monitor interactions between migrating glioma cells and nerve fibers.

Glioma-cell migration is usually assessed in dissociated cell cultures, spheroid cultures, acute brain slices and intracranial implantation models. However, the interactions between migrating glioma cells and neuronal tracts remain poorly understood. We describe here a protocol for the coculture of glioma cells with myelinated axons in vitro. Unlike other methods, this protocol allows the creation of in vitro conditions that largely mimic the complex in vivo environment. First, long retinal axons from embryonic chicken are formed in an organotypic culture. Glioma cells are then positioned in the vicinity of the explants to allow them to contact the axons, interact with them and eventually migrate along them. High-resolution video microscopy and confocal microscopy can be used to monitor the migratory behavior. This protocol, which takes about 5 days to complete, could be applied to different types of tumor cells that interact with neurites, and is suitable for pharmacological and genetic approaches aimed at elucidating mechanisms underlying tumor migration.

Reactive oxygen species-mediated regulation of eNOS and iNOS expression in multicellular prostate tumor spheroids.

Nitric oxide (NO) generated by either endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS) may be involved in prostate tumorigenesis through the inhibition of reactive oxygen species (ROS)-induced apoptosis. Multicellular DU-145 prostate tumor spheroids endogenously generated NO that paralleled the production of ROS. With increasing spheroid size, eNOS expression was downregulated, whereas an upregulation of iNOS expression was observed. In parallel, NO generation declined, as evaluated by the NO indicator diaminofluorescein-2 diacetate (DAF-2DA), suggesting that NO generation in DU-145 tumor spheroids is mainly mediated by eNOS. Elevation of ROS by treatment of tumor spheroids with either buthionine sulfoximine (BSO) or hydrogen peroxide resulted in upregulation of eNOS, whereas iNOS was downregulated. Furthermore, eNOS expression was increased by epidermal growth factor (EGF) in a redox-sensitive manner. Upregulation of eNOS after treatment with hydrogen peroxide was apparently transduced through receptor tyrosine kinase signaling pathways since it was abolished by the protein kinase C (PKC) inhibitor bisindolylmaleimide-1 (BIM-1), the p21(ras) inhibitor S-trans-trans-farnesylthiosalicylic acid (FTS), the c-Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1/2 activation. Endogenous NO may serve to escape from oxidative stress-induced apoptosis since treatment of tumor spheroids with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO) as well as the NO synthase inhibitor N-omega-amino-L-arginine (L-NAA) increased cleaved caspase-3. Consequently, lowering intracellular NO levels with either L-NAA or PTIO significantly raised ROS levels, indicating that endogenously generated NO may play a role as a ROS scavenger, thereby protecting exponentially growing tumor spheroids from ROS-induced apoptosis.