Thesis (doctoral)--Rijksuniversiteit te Utrecht.

Thesis (doctoral)--Rijksuniversiteit te Utrecht.

Cells respond to chemical, mechanical and topographical cues both in vivo and in vitro. Much research has been carried out into the effects of chemical signals and to a lesser degree, mechanical. However, less is known about cell responses to topographical cues, particularly to topographies with nanoscale dimensions. Understanding how cells respond to topography is of particular interest to the field of tissue engineering, where it is crucial to characterise the effects that biomaterial surfaces have on the cells that they come into contact with. Observations of the impact that topographic signalling has on cells, within two tissue engineering systems, are discussed in this thesis. These systems are: polymer conduits for peripheral nerve regeneration and thin films for the replacement of the retinal pigment epithelium. Understanding the effects that micro and nano scaled topographies have on nerve and retinal cell regeneration is important for successful development and implementation of appropriate tissue engineered devices. In order to fabricate topographical patterns on biomaterial surfaces, a number of fabrication techniques were investigated. The fundamental requirement of these techniques was for reliable production of uniform nano and micro scale topographical patterns over large lateral areas (millimeter scale). Initially, the suitability of electrohydrodynamic lithography (EHDL) was assessed. EHDL is a relatively new technique, first published in 2000, which employs electrostatic forces to pattern thin polymer films. Subsequently, techniques traditionally associated with the computing industry, such as e-beam lithography and reactive ion etching, were evaluated. Following successful pattern fabrication, NG108-15 and ARPE-19 cells were cultured on grooved topographies. Against a baseline parameter of elapsed time, the cell morphologies and their propensity for alignment with the grooves was rigorously assessed and compared. ARPE-19 and NG108-15 cell responses differed from one another, and were sensitive to varying groove dimensions. Ultimately, the developing morphologies (for both cell types) proved to be clearly dependent on groove dimensions and elapsed time.

A 7-9 fold enriched preparation of bovine retinal pigment epithelial cell plasma membrane was prepared and characterized by enzymatic analysis. SDS-polyacrylamide gel electrophoresis and transmission electron microscopy revealed a large rod outer segment contamination in the preparation due to a tight retinal pigment epithelial cellular adhesion to the rod photoreceptor cells. The contaminating rhodopsin was partially removed by anti-rhodopsin immunoaffinity chromatography as determined by SDS-polyacrylamide gels stained with coomassie blue or silver. Monoclonal antibodies raised against the retinal pigment epithelial plasma membrane preparation cross reacted with rod outer segment preparations. A monoclonal antibody, designated Rho-5A3, was classified as an IgG₃ kappa light chain immunoglobulin. It was shown to be specific for rhodopsin as determined by radioimmune labeling of bovine rod outer segment membrane proteins electrophoretically transferred to CNBr-activated paper. Limited proteolytic digestion of rhodopsin followed by electrophoretic transfer to CNBr- activated paper localized the binding site of this antibody to the N-terminal two-thirds of the rhodopsin molecule. Competition assays with rhodopsin polypeptides further defined the antigenic site to be within the 17-39 amino-acid segment of rhodopsin. The Rho-5A3 antibody did not bind to sealed ROS discs or frozen-thawed ROS discs but did bind to Triton X-100 solubilised discs indicating a detergent solubilisation dependence for antigenic site accessibility. Cultures of bovine retinal pigment epithelial cells were started by initial enzymatic isolation followed by recovery in RPMI-1640 culture medium. The retinal pigment epithelial cells established a doubling time of 52 hours until the cells reached culture confluency . The cells also maintained many of their in vivo characteristics such as a high degree of pigmentation and an abundance of microvilli. Cell surface glycoproteins labelled with FITC-Con A, FITC-WGA, and FITC-RCA showed dense and random surface labelling patterns. Fluorescent labels were induced to redistribute to central spots and clear from the cell surface by incubating the labelled cells in buffer for 60 minutes at 37°C. Treatment by the appropriate saccharide inhibitors indicated that the labelled sites had undergone endocytosis by the cell. Continuous labelling experiments indicated that redistribution and internalization is constantly occurring so that previously unlabeled receptors become accessible for labelling. As a result a dense pattern of label on the cell surface was maintained. The protein actin, with apparent Mr =46,000, was detected with rabbit anti-actin antisera labelling of the RPE plasma membrane preparation proteins electrophoretically transferred to nitrocellulose paper. Immunofluorescent labelling using the rabbit anti-actin antisera confirmed biochemical studies that actin was a major component of the bovine RPE cell. The activation of actin filaments may play an important role in the phagocytosis of bovine rod outer segments by retinal pigment epithelial cells in tissue culture. Scanning electron microscopy and transmission electron microscopy have shown that 2 week old bovine retinal pigment epithelial cells in vitro can be induced to recognize, attach, and engulf dark adapted sealed rod outer segments. In summary a monoclonal antibody was raised against a bovine retinal pigment epithelial cell plasma membrane preparation, however, the antibody raised proved to be specific for rhodopsin, a contaminating ROS protein found in the preparation. The monoclonal antibody, designated Rho-5A3, was fully characterized and its antigenic site determined. Finally, bovine retinal pigment epithelial cells grown in tissue culture acted as a model system for studying cell surface components and rod outer segment phagocytosis at the levels of fluorescence, scanning electron microscopy, and transmission electron microscopy. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

INTRODUCTION: Optical coherence tomography (OCT) is used to study retinal structure in schizophrenia. Changes in retinal structure, especially the retinal nerve fiber layer (RNFL) have been correlated with psychotic disorders. Along with a decrease in macular outer nuclear layer (ONL) thickness and an increase in macular outer plexiform layer (OPL). However, measurement variability is a concern for inner retinal layers since there are various generations of OCT devices resulting in differing measurements. We investigated the inter- and intra-device agreement of macular thickness between spectral domain (SD-OCT) and swept source-OCT (SS-OCT), and compared macula and peripapillary group differences in schizophrenia using SS-OCT for inner retinal layers. Additionally, we expanded on our previous work and investigated whether baseline outer retinal layer data for macular ONL and OPL thickness predicted clinical and cognitive changes in individuals with schizophrenia. METHODS: For the inter- and intra-scanner study, macular OCT thickness was obtained for schizophrenia (SZ, n=30) and healthy controls (HC, n= 22) subjects using SD-OCT (Heidelberg Spectralis) and SS-OCT (DRI Topcon Triton). Peripapillary thickness was obtained using SS-OCT. RNFL, ganglion cell-inner plexiform complex (GCL+), RNFL+ GCL+ (GCL++), and macular thickness were collected. For the longitudinal study, 7 participants diagnosed with either schizophrenia or schizoaffective baseline OCT measurements and clinical measures were obtained for all 7 participants from study 1, along with 6 months follow up clinical measures. OCT measurements for the macular OPL and ONL were gathered using the Heidelberg Spectralis Clinical and cognitive data was gathered. All statistical analyses were performed using R software. RESULTS: There was excellent inter-scanner agreement for GCL+ and GCL++ with Interclass correlation coefficient (ICC) values between r =0.92-0.99. Good to excellent intra-scanner agreement was present except for macular RNFL in the SS-OCT device. No significant peripapillary group differences were identified. Poorer (Global Assessment of Functioning) GAF scores were correlated with thinner macular layers. Greater mania symptoms were associated with smaller peripapillary GCL+ thickness (r=-0.43, p=0.03). Poor overall cognition was associated with smaller peripapillary retinal thickness (r=0.36, p=0.02). For the longitudinal study, an increase in baseline OPL thickness was correlated with worse positive symptoms according to the Positive and Negative Symptom Severity(PANSS) at the 6 month follow up (r=0.77, p=0.04) with a trend level effect for PANSS total scores (r=0.71, p=0.08). There was no significant correlation between the change in clinical or cognitive outcomes for 6 months and baseline OPL and ONL thickness. CONCLUSION: While there is RNFL variability, GCL+ and GCL++ are comparable between scanners SD-OCT and SS-OCT. Given that RNFL thinning is strongly implicated in psychotic disorders, the use of OCT scanners should not be interchanged due to increased RNFL measurement variability. Additionally, though further research is needed on investigating changes in clinical outcomes with changes in OCT measurements, OPL thickness might be a predictor of long-term clinical outcomes or changes in brain pathology for individuals with schizophrenia.

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Recent advances in retinal imaging have made it possible to take measurements of retinal oxygen saturation noninvasively in humans. This allows studying the supply of oxygen in healthy and diseased retinae, thereby advancing our understanding of both the normal functioning of the retina and of retinal pathologies. However, retinal oximetry is still a research tool only and requires further improvement before being used in a clinical setting. Here, a single-wavelength flickering light was used to increase retinal blood flow in healthy subjects. This increase is revealed by both vasodilation and an increase in retinal oxygen saturation. A flickering light stimulus provides the means to assess the sensitivity of any retinal oximetry system, as such systems should be able to pick up this increase in retinal blood flow. In addition, the flickering light allows for com- parison to be made within rather than between subjects and can be used to examine the activation of the eye. This reduces the influence of potential confounding factors between subjects including differences in fundus pigmentation and illumination. The most commonly used method to measure retinal oxygenation is the optical density ra- tio (ODR) approach. The standard approach is to compute the average ODR for each vessel segment by combining the hundreds of individual ODR readings and then to use the mean of these segment averages as a measure of oxygen saturation. Alternatively, it has been suggested that the peak location of Gaussian functions fitted to histograms of individual ODR readings can be used as an measure of retinal oxygenation. In response to a 10Hz flickering light, the venular diameter increased by 3.44% (SEM: ±0.53%) (n=16, p<0.05) and the arteriolar diameter by 1.87% (±0.72 %) (p<0.05). The optical density ratio, measured with the Gaussian fit, decreased in the venules from 0.713 (±0.015) to 0.694 (±0.015) (p<0.05). No changes in arteriolar optical density ratios were measured. The post-flicker measurement was computed as the average of up to four post-flicker datasets obtained at 10s, 20s, 30s and 40s after onset of flickering. These results suggest that the flickering light increased retinal blood flow. The mean absolute percentage error was lower in venules for the Gaussian fit method than for the gold standard method for datasets taken at 30s and 40s after onset of flickering. Thus, the Gaussian fit method was more robust. All measurements were taken with a custom-made retinal oximeter. The pixel intensity of the blood vessel and the intensity on either side of the vessel had to be extracted to compute the individual optical density ratios. This required the automatic extraction of the retinal vasculature. Two such algorithms were developed and applied to two databases of retinal fundus images: the DRIVE and the novel DR HAGIS database. One algorithm was purely based on the pixel intensities, while the other made use of oriented Gabor filters. These two algorithms segmented the images to a similar accuracy (DRIVE: 94.56% and 94.54%, DR HAGIS: 95.83% and 95.71% for the intensity and Gabor filter based algorithm, respectively) and performed as well as a human expert (DRIVE: 94.73%). These algorithms were of sufficient quality to extract individual segments for the oximetry study and to align fundus images.

PURPOSE: This study aims to quantify the retinal vascular blood flow in eyes affected by unilateral central retinal vein occlusions (CRVO) or branch retinal vein occlusions (BRVO). We created and explored a new, unitless metric for the severity of these diseases: relative blood flow (RBF). We then contextualized RBF in terms of patient demographics, ocular presentation and other systemic conditions, as well as explored its efficacy as a predictor of future outcomes. METHODS: Data was collected from 20 control subjects and 32 patients with clinically diagnosed retinal vein occlusions (15 CRVO and 17 BRVO). Laser speckle flowgraphy was then used to quantify retinal vascular blood flow in terms of mean blur rate, a metric shown to be highly heterogeneous between patients but fairly consistent in intra-patient repeated measurements over time. After confirming this and establishing a strong correlation between a healthy patient’s two eyes, we used an RVO patient’s fellow eye as a nondiseased expectation and presented relative blood flow as the ratio between their diseased and healthy eye. We then correlated this data with demographic variables and disease characteristics from patients’ medical history. RESULTS: We found an average blood flow decrease of 26% in CRVO eyes relative to healthy eyes in the same patients and an average decrease of 7% in BRVO eyes. In CRVO, duration of occlusion, central macular thickness, intraocular pressure, diabetes, previous laser and injection treatments, and an injection within three months after blood flow measurement were significantly associated with relative blood flow. In BRVO, no demographic variables or disease characteristics were significantly associated with relative blood flow. CONCLUSIONS: Relative blood flow represents a promising new, consistent and informative metric for quantifying the severity of unilateral retinal vein occlusions. With both descriptive and predictive properties in eyes with CRVO, future work should explore its great potential.

Purpose. These experiments were designed to characterize tight junction formation by retinal pigment epithelial (RPE) cells in vitro and to compare the effects on this process of hormonally defined medium (HDM) and serum- containing medium. Methods. Formation of RPE tight junctions was analyzed in freshly isolated rat RPE cells maintained either in HDM or serum-containing medium. Junctions were evaluated functionally by measuring transepithelial electrical resistance (TER) and permeability and structurally by immunolocalization of the junction-associated actin microfilaments. Calcium dependency of the junction was determined by reducing media calcium concentration. Results. RPE cells cultured in serum-free HDM developed calcium-dependent tight junctions, which exhibited TER levels > 150 Ω · cm 2 and low paracellular permeability. Serum-containing media inhibited tight junction formation as indicated by significant reductions in TER and increases in permeability. Junction-associated actin microfilaments and cell density were unchanged. Conclusions. Tight junction formation by RPE cells is inhibited by serum. This activity may play an important role in responses of the RPE layer to injury, contributing to the pathologic progression of blood- retinal barrier dysfunction.