Volker Enzmann

Macular Thickness Measurements in Healthy Eyes Using Six Different Optical Coherence Tomography Instruments

PURPOSE To compare central retinal thickness (CRT) measurements in healthy eyes by different commercially available OCT instruments and to compare the intersession reproducibility of such measurements. METHODS Six different OCT instruments (Stratus OCT [Carl Zeiss Meditec, Inc. Dublin, CA], SOCT Copernicus [Reichert/Optopol Technology, Inc., Depew, NY], Spectral OCT/SLO [Opko/OTI, Inc., Miami, FL], RTVue-100 [Optovue Corp., Fremont, CA], Spectralis HRA+OCT [Heidelberg Engineering, Inc., Heidelberg, Germany], and Cirrus HD-OCT [Carl Zeiss Meditec, Inc.]) were used to assess CRT in both eyes of healthy subjects. Measurements were performed in two different sessions on the same day with each of the systems. From these measurements, the mean CRT was calculated. For the assessment of the intersession reproducibility of the instruments, we calculated the coefficient of the variation of test-retest variation. RESULTS Twenty healthy subjects were included in the study. Compared with the Stratus OCT all spectral OCT instruments showed significantly higher CRTs. The Spectralis HRA+OCT and Cirrus HD-OCT showed similar CRT values but significantly higher values than did all other instruments. The coefficients of variation for repeated measurements was 3.33% for the Stratus OCT, 0.46% for the Spectralis HRA+OCT, 3.09% for the Cirrus HD-OCT, 2.23% for the OCT/SLO, 2.77% for the RTVue-100 OCT, and for the SOCT 3.5%, respectively. discussion. The six OCT systems provided different results for CRT. The measurements with the Stratus OCT showed the lowest thicknesses, whereas those with the Cirrus HD-OCT and Spectralis HRA+OCT yielded the highest ones. These discrepancies can be explained by the differences in the retinal segmentation algorithms used by the various OCT systems. Whereas the Spectralis HRA+OCT and Cirrus HD-OCT include the RPE layer in the retinal segmentation, the other instruments do not. The data imply that the different OCT systems cannot be used interchangeably for the measurement of macular thickness.

Morphologic Changes in Patients with Geographic Atrophy Assessed with a Novel Spectral OCT-SLO Combination

PURPOSE To investigate the appearance of geographic atrophy in high-resolution optical coherence tomography (OCT) images, the fundus autofluorescence (FAF) pattern, and infrared images simultaneously recorded with a novel combined OCT-scanning laser ophthalmology (SLO) system. METHODS Patients aged over 50 years with geographic atrophy secondary to dry age-related macular degeneration (ARMD) were assessed in a prospective cross-sectional study by means of simultaneous spectral OCT-SLO (Spectralis HRA+OCT; Heidelberg Engineering, Heidelberg, Germany). The integrity of the retinal layers was analyzed in the apparently normal areas, the junctional zone between the normal retina and the geographic atrophy, and the atrophic area. The presence and integrity of the external limiting membrane, the photoreceptor inner segments, the outer segments, and the retinal pigment epithelium were assessed. RESULTS Fifty-two eyes of 52 patients (28 women, 24 men) aged 51 to 92 years were examined. Retinal layer alterations were documented, not only in atrophic zones, but also in junctional zones surrounding the geographic atrophy. Disintegration of the retinal layers began in the RPE and adjacent retinal layers, such as the photoreceptor inner and outer segments and external limiting membrane. CONCLUSIONS Novel imaging modalities will provide further valuable insight into ARMD pathogenesis. The key to understanding the morphologic change lies in in vivo depiction of retinal layers by spectral OCT technology in combination with other imaging modalities such as FAF.

Stem Cell-Based Therapeutic Applications in Retinal Degenerative Diseases

Retinal degenerative diseases that target photoreceptors or the adjacent retinal pigment epithelium (RPE) affect millions of people worldwide. Retinal degeneration (RD) is found in many different forms of retinal diseases including retinitis pigmentosa (RP), age-related macular degeneration (AMD), diabetic retinopathy, cataracts, and glaucoma. Effective treatment for retinal degeneration has been widely investigated. Gene-replacement therapy has been shown to improve visual function in inherited retinal disease. However, this treatment was less effective with advanced disease. Stem cell-based therapy is being pursued as a potential alternative approach in the treatment of retinal degenerative diseases. In this review, we will focus on stem cell-based therapies in the pipeline and summarize progress in treatment of retinal degenerative disease.

Comparison of electrically evoked cortical potential thresholds generated with subretinal or suprachoroidal placement of a microelectrode array in the rabbit

The aim of the study was to directly compare the threshold electrical charge density of the retina (retinal threshold) in rabbits for the generation of electrical evoked potentials (EEP) by delivering electrical stimulation with a custom-made microelectrode array (MEA) implanted into either the subretinal or suprachoroidal space. Nine eyes of seven Dutch-belted rabbits were studied. The electroretinogram (ERG), visual evoked potentials (VEP) and EEP were recorded. Electrodes for the VEP and EEP were placed on the dura mater overlying the visual cortex. The EEP was recorded following electrical stimulation of the MEA placed either subretinally beneath the visual streak of the retina or in the suprachoroidal space in the rabbit eye. An ab externo approach was used for placement of the MEA. Liquid perfluorodecaline (PFCL; 0.4 ml) was placed within the vitreous cavity to flatten the neurosensory retina on the MEA after subretinal implantation. The retinal threshold for generation of an EEP was determined for each MEA placement by three consecutive measurements consisting of 100 computer-averaged recordings. Animals were sacrificed at the conclusion of the experiment and the eyes were enucleated for histological examination. The retinal threshold to generate an EEP was 9 +/- 7 nC (0.023 +/- 0.016 mC cm(-2)) within the subretinal space and 150 +/- 122 nC (0.375 +/- 0.306 mC cm(-2)) within the suprachoroidal space. Histology showed disruption of the outer retina with subretinal but not suprachoroidal placement. The retinal threshold to elicit an EEP is significantly lower with subretinal placement of the MEA compared to suprachoroidal placement (P < 0.05). The retinal threshold charge density with a subretinal MEA is well below the published charge limit of 1 mC cm(-2), which is the level below which chronic stimulation of the retina is considered necessary to avoid tissue damage (Shannon 1992 IEEE Trans. Biomed. Eng. 39 424-6).

Decreased Visual Function after Patchy Loss of Retinal Pigment Epithelium Induced by Low-Dose Sodium Iodate

PURPOSE To correlate damage to the retinal pigment epithelium (RPE) with decreased visual function after the systemic administration of sodium iodate (NaIO(3)). METHODS Damage was produced in mice by injection of 15, 25, or 35 mg/kg NaIO(3). Visual function was assessed with the cued water maze (WM) behavioral test and the optokinetic reflex (OKR) measurement at different times after injection. Autofluorescence in whole eye flatmounts was quantified, and hematoxylin and eosin staining of paraffin sections was performed to assess changes in the outer retina. RESULTS After 15 mg/kg NaIO(3), cued WM test results were normal, whereas OKR measurements were significantly decreased at all times. Focal RPE loss began on day 21, but no significant damage to the outer nuclear layer was observed. After 25 mg/kg NaIO(3), the cued WM test was transitionally reduced and the OKR measurement again decreased at all times. Large areas of RPE loss occurred on day 14 with a reduced outer nuclear layer on the same day. With 35 mg/kg NaIO(3), the cued WM test was reduced beginning on day 14 with complete obliteration of the OKR beginning on day 3, large areas of RPE loss on the same day, and a reduced outer nuclear layer on day 7. CONCLUSIONS Stable, patchy RPE loss was observed with a low concentration of NaIO(3). The OKR measurement showed changes in visual function earlier than the cued WM test and before histologic findings were observed.

Stem cells as tools in regenerative therapy for retinal degeneration

OBJECTIVE To describe the use of stem cells (SCs) for regeneration of retinal degenerations. Regenerative medicine intends to provide therapies for severe injuries or chronic diseases where endogenous repair does not sufficiently restore the tissue. Pluripotent SCs, with their capacity to give rise to specialized cells, are the most promising candidates for clinical application. Despite encouraging results, a combination with up-to-date tissue engineering might be critical for ultimate success. DESIGN The focus is on the use of SCs for regeneration of retinal degenerations. Cell populations include embryonic, neural, and bone marrow-derived SCs, and engineered grafts will also be described. RESULTS Experimental approaches have successfully replaced damaged photoreceptors and retinal pigment epithelium using endogenous and exogenous SCs. CONCLUSIONS Stem cells have the potential to significantly impact retinal regeneration. A combination with bioengineering may bear even greater promise. However, ethical and scientific issues have yet to be solved.

An improved MTT assay using the electron-coupling agent menadione

A modification of the MTT based tetrazolium colorimetric assay is described. Using the electron-coupling agent menadione formazan formation by murine splenocytes and P-815 cells was significantly increased whereas dye reduction by macrophages was hardly influenced. These observations suggest that it should be possible to improve the tetrazolium based cytotoxicity assays of murine macrophages against cells of the syngeneic tumour cell line P-815.

Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin(-)CD105(+)) were harvested from GFP(+)-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP(+)-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4 days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.

Mechanisms of Graft Rejection in the Transplantation of Retinal Pigment Epithelial Cells

PURPOSE The role of activated retinal pigment epithelium (RPE) cells was investigated in the rejection after subretinal transplantation. METHODS RPE cells from 7 pigmented rabbits were separated and evaluated regarding their MHC class II expression as the sign of activation. The activation of the RPE cells was augmented with a treatment of 1,000 U/ml interferon gamma (IFN-gamma) for 8 days. These cells were then transplanted into 7 albino rabbits. As control, RPE transplantations without a pretreatment were performed in 7 albino rabbits. Six weeks after the transplantation, the transplanted eyes were enucleated and histology was performed. RESULTS In culture, without IFN-gamma addition, 11.38 +/- 0.94% of the RPE cells presented MHC class II. After IFN-gamma treatment, this quantity increased to 78.26 +/- 1.46% of the RPE cells. These cells transplanted into the rabbits caused an obvious rejection in the transplantation area which was verified histologically. The control group presented a transplantation area without signs of rejection or inflammation. CONCLUSION In culture, some of the adult RPE cells are activated. These cells may accelerate the rejection cascade after transplantation. An elimination of activated RPE cells from the transplant should be recommended before transplantation.

Retinal pigment epithelial cells activate uveitogenic T cells when they express high levels of MHC class II molecules, but inhibit T cell activation when they express restricted levels

The retinal pigment epithelium (RPE) plays an important role in maintaining the privileged status of the eye. In this study, we examined the outcome of the in vitro interaction between uveitogenic T cells and non-activated or activated RPE cells. An interphotoreceptor retinoid-binding protein (IRBP)-reactive T cell line was established and RPE cell monolayers prepared. The expression of MHC class II molecules on RPE cells activated by graded doses of IFN-gamma was determined by flow cytometry. Proliferation and cytokine production of IRBP-reactive T cells were measured after exposure to IFN-gamma-activated RPE cells. We found that RPE cells expressed various levels of MHC class II molecules depending on the concentration of IFN-gamma used. IRBP-specific T cells could be stimulated or inhibited by exposure to RPE cells. The dual effect of RPE cells might be regulated via different levels of MHC class II molecules on the RPE cells. It suggests that controlling the immune activities of uveitogeneic T cells by varying the levels of MHC class II expression on the RPE might be one of the regulatory mechanisms by which the RPE maintains immune privilege in the eye.