Poster Sessions Saturday

Background: Retinal microglia (MG) are cells of the innate immune system involved in tissue homeostasis, inflammation and wound healing in the eye. They are complemented by recruited macrophages (Mac) from the blood during disease and inflammation when the blood-retina barrier is breached. The origin, turnover and function of resident MG versus blood-derived Mac however, remain ill-defined due to a lack of cell-specific in vivo systems. Methods: The origin of retinal MG was investigated by pulse-labeling approaches targeting the embryonic primitive hematopoiesis and fate mapping of definitive hematopoiesis using immunohistochemistry (IHC) and flow cytometry (FACS) in novel transgenic reporter mice. In addition, we investigated the turnover of resident MG in the steady state using MG-specific CX₃CR1-Cre-ER reporter mice and parabiotic mice by IHC and FACS. Finally, we explored the relative distribution and function of resident microglia cells and infiltrating macrophages in a model for laser-induced CNV by in vivo imaging and IHC. Results: Our study demonstrates for the first time that retinal microglia cells are uniquely derived from primitive hematopoiesis. We found that MG are not supplemented by blood-borne myeloid cells but are instead self-maintained. In the laser-induced CNV model we found that both resident MG and blood-derived macrophages accumulate at sites of laser injury and precede the development of CNV. Conclusion: We provide a new in vivo system which for the first time enables us to determine the embryonic origin and turnover of retinal MG. This model allows us to discriminate between resident MG and infiltrating Macs in the steady state and under pathological conditions and to better understand the cellular and molecular mechanisms underlying CNV development.

Fragestellung: Bei der altersbedingten Makuladegeneration (AMD) findet eine Reihe von noch unzureichend erforschten pathologischen Prozessen statt. Über die Rolle der Mikroglia bei der AMD gibt es bislang nur begrenzte Kenntnisse. Durch die Anwendung von Mikroglia-hemmenden Verbindungen sollte deshalb überprüft werden, inwiefern sich die Aktivität und Migration der Mikroglia in den neovaskulären Bereichen und dadurch das Ausmaß einer choroidalen Neovaskularisation (CNV) beeinflussen lassen. Methodik: Augen von adulten Wildtyp und CX3CR1^GFP/+-Mäusen wurden mit einem Argonlaser behandelt, um eine CNV zu induzieren. Nach der Laserbehandlung wurden Lösungen von Minocyclin, Hydrocortison sowie der beiden Peptide TKP und RGDS intravitreal injiziert. Mit PBS injizierte Tiere dienten als Kontrolle. 2, 4 und 7 Tage nach der Laserbehandlung wurde eine in-vivo-Bildgebung durchgeführt. Anschließend wurden die Augen entnommen und Gefrierschnitte hergestellt. Die Mikroglia wurde unter Verwendung des CD11b-Antikörpers gefärbt. Doppelfärbungen von Mikroglia wurden gegen pro-angiogene Faktoren, inflammatorische Cytokine und Rezeptoren durchgeführt. Die Migration der Mikroglia und das Ausmaß der Neovaskularisation wurden anhand der erhöhten Fluoreszenz in den Laserherden der CX3CR1^GFP/+-Mäuse und mittels ICG-Angiographie quantifiziert. Ergebnisse: Anhand der relativen Fluoreszenz zeigte sich an den Tagen 2 und 4 nach Laserbehandlung, dass in den mit Minocyclin und TKP behandelten Tieren signifikant weniger Mikroglia-Zellen in die Laserherde migriert hatten (p< 0,001 für Minocyclin und p< 0,05 für TKP). Im Bereich der Laserherde konnte 4 Tage nach der Laserbehandlung bei den Tieren, die mit Minocyclin oder TKP behandelt wurden, ein signifikant verringertes Ausmaß der CNV festgestellt werden (p=0.01 bzw. p=0.03), und 7 Tage nach der Behandlung war das Ausmaß der CNV immer noch tendenziell geringer. Die Immunreaktivität der CD11b-positiven Zellen für FGF-2, IL-6 und TGF-β war unter dem Einfluß von Minocyclin und TKP an den Tagen 4 und 7 nach der Laserbehandlung teilweise deutlich verringert. Schlussfolgerung: Nach der Applikation der Mikroglia-hemmenden Substanzen Minocyclin und TKP zeigte sich im Modell der laserinduzierten CNV eine deutliche Verringerung der Migration der Mikroglia in die Laserherde, ein verringertes Ausmaß der CNV und eine Verringerung der Expression bestimmter pro-angiogener und pro-inflammatorischer Faktoren.

Introduction: Microglial cells are the intrinsic immune cells of the central nervous system and of the retina. Involvement of microglial cells in the pathology of CNV became of increasing interest. One critical point is to investigate the occurrence and the role of various receptors that may influence the behaviour of microglial cells. One family of such receptors are purinergic receptors that respond to ATP and initiate various processes in the microglial cells. We here report on four important purinergic receptors and their inhibitor PPADS in the mouse model of laser-induced CNV. Methods: Mice were treated with a laser to induce CNV and divided into four groups. One group received no subsequent treatment, and eyes were isolated at 1, 4, 7 and 14 days after laser treatment. Mice of the second, third and fourth group received intravitreal injection of 2 µl PPADS or PBS immediately after laser treatment or eye drops of PPADS once per day after laser treatment until the third day, respectively. Mice received fluorescence angiography examination at 4 days after laser treatment, and eyes were isolated to prepare cryosections for immunohistochemistry. Microglial cells were stained using CD11b antibody. Double staining of microglial cells was performed against purinergic receptors P2X4, P2X7, P2Y2 and P2Y12. We checked immunohistochemically the presence of purinergic receptors as well as some typical factors on microglial cells. Results: In the normal mouse retina, microglial cells show almost no immunoreactivity for the investigated purinergic receptors. After laser treatment, immunoreactivity for the purinergic receptors increases clearly, with the maximal response 4 days after laser treatment. After treatment with the PPADS, immunoreactivity for P2X4, P2X7 and P2Y2 is clearly decreased, and there were less microglial cells positive for the purinergic receptors compared to PBS injection group. There were less percent of microglial cells positive for TGFb-1, VEGF and VEGFR2 after PPADS treatment in the laser spot, but more percent of microglial cells positive for PDGF-b and VEGFR1 comparing with PBS injection group. Conclusion: Microglial cells express purinergic receptors P2X4, P2X7, P2Y2 and P2Y12 in laser-induced CNV, especially at 4 days after laser treatment. PPADS inhibits the expression of P2X4 and P2X7. Topical or intravitreal application of PPADS reduces the reactivity of microglial cells as well as the leakage of fluorescence from CNV.

The pentacyclic triterpenoid, betulinic acid, was recently shown to reduce cerebral ischemia-reperfusion injury in mice. The purpose of the present study was to test the hypothesis that betulinic acid protect against ischemia-reperfusion injury in the mouse retina. Ocular ischemia was induced in mice by increasing intraocular pressure to 110 mm Hg for 45 min by infusion of balanced salt solution via a cannula located in the anterior chamber. The other eye of the same animal, in which intraocular pressure was elevated to a physiological level of 15 mm Hg, served as control. One goup of mice received placebo and the other group betulinic acid (50mg/kg/day p.o. once daily starting one day before and ending seven days after increasing intraocular pressure). After seven days, the animals were killed and retinal wholemounts were prepared and stained with cresyl blue. Neurons in the retinal ganglion cell layer were counted. In addition, redox gene expression was determined in isolated retinas by quantitative PCR. Ischemia-reperfusion markedly reduced neuron number in the retinal ganglion cell layer of placebo-treated mice. In contrast, only an insignificant reduction in neuron number was observed in betulinic acid-treated mice. In mice treated with betulinic acid, the antioxidative enzymes superoxide dismutase 1, superoxide dismutase 3 and heme oxygenase 1 were upregulated in the retina. In contrast, mRNA expression was not changed for the three nitric oxide synthase isoforms, hypoxia-inducible factor-1alpha or vascular endothelial growth factor. In conclusion, our data suggest that betulinic acid protects from ischemia-reperfusion injury in the mouse retina by reducing oxidative stress via superoxide dismutase 1, superoxide dismutase 3 and heme oxygenase 1 upregulation.

Purpose: Sphingolipids are multifunctional lipid mediators. The role of these lipids has been established in cellular functions such as cell survival, growth, apoptosis, inflammation and angiogenesis. Sphingosine-1-phosphate (S1P) is synthesized by two sphingosine kinase isoforms (Sphk1 and Sphk2) with different subcellular distribution. S1P is an important operator in retinal neovascularisation. The roles of Sphk2 are complex, its functions in pathological angiogenesis are not well characterized and its dysfunction has been shown to facilitate the development of various disease states. Here, we investigated for the first time the role of Sphk2 in vascular diseases of the retina. Material and Methods: The oxygen-induced retinopathy model is used as a pathological angiogenesis model. Neonatal C57BL/6J and EllaCrexJos5a (Sphk2 overexpressing mice) with a C57BL/6J background are subjected to 75% oxygen for 5 days, from postnatal day 7 (P7) to P12 and then returned to room air. Immunhistochemistry on wholemount and paraffin embedded retina slides were performed and neovascularisation was characterized. Results: Sphk2↑ mice that were not subjected to the disease model show a reduction in their ONL and INL thickness, especially at P12. Furthermore more deep thick blood vessel are found in Sphk2↑. In the OIR model, the blood vessel loss in the early phase (P7-P12) was significantly decreased in Sphk2↑ mice (14.96 % reduction, ***P< 0.0001). At the later states the neovascularisation occurs earlier and at P17 the new blood vessel system shows a more chaotic organization of the blood vessel system. In this state, SphK2↑ show more GFAP-positive Müller cell processes than wildtype mice, but glutamine synthetase stainings, highlighting Müller glia cell bodies and processes, do not show morphologic changes of enhanced gliosis. Conclusion: Our results show that Sphk2 plays a role in the development of the retina and its vascular structure under normal and pathophysiologic conditions. We found reduced vessel loss in the initial stage of the disease model followed by increased neovascularisation with more pathologic vessel formation. While the cellular and molecular mechanisms yet have to be unravelled, inhibition of Sphk2 may offer novel strategies in the treatment of neovascular diseases of the posterior segment of the eye.

Purpose: To establish the role of proper retinal vascularization as neuroprotective factor in the presence of diabetic retinopathy. Methods: We modelled diabetes mellitus in Chinchilla rabbits by dithizone, in Wistar rats and CBA/C57B1xK/F1 mice by streptozotocine. Observing period did not exceed 6 months. Enucleated eyes were studied by histological methods. Results: We revealed no significant changes in the structure of the retinas of mice and rats by observing the animals from 2 to 6 months. In the eyes of both intact and diabetic rats and mice we observed an extencive rete of blood vessels in all retinal inner layers during all periods of observation. In the outer retinal layers there were no blood vessels, and its trophic processes provided by the choroid. We determined (by using ordinary histological staining (hematoxylin-eosin)) destruction of the photoreceptor layer, diminishing of neurons quantity in nuclear layers, diminishing of neurons in ganglion cell layer, thinning of the reticular layers, proliferation of glial elements, destruction of the pigment epithelium and focal retinal atrophic changes in retinas of diabetic rabbits after 16-17 weeks of observation. Rabbits retina is almost deprived of proper retinal blood vessels in all its layers, excluding only medullated fiber. Theese vessels are located nasally and temporally from the optic disc both in intact and diabetic rabbits. However, they supply with blood only medullated fiber. The almost whole retina is supplied from choroid. This feature of retinal blood supply in rabbit makes retina particularly sensitive to damaging factors including diabetogenic. This phenomenon, against the background of early free-radical and PAS-positive substances damage (that we have previously established) increase the retinal metabolism imbalance in diabetic rabbits. All of these diminished retinal ability to resist neurodegeneration development. Conclusion: Proper retinal vascularization (as in the eyes of rats and mice) is one of the natural neuroprotective factor, continuing acting at the presence of diabetes mellitus.

Background and purpose: Retinal neovascularization is a blinding complication in diabetic retinopathy, wet age-related macular degeneration, retinopathy of prematurity, and after central retinal vessels occlusion. Despite significant advances in traditional treatment we could not restore lost visual functions to date. Successful results and broad application prospects of cryopreserved stem cells in different areas of medicine sparks interest in their use in ophthalmology. The effect of cryopreserved cord blood derived nuclear cells (CCBNC) on neovascular retinopathy (NVR) in experimental rats was studied. Materials and methods: In our study we used newborn Wistar rats (n=40). Brinzolamide-induced retinopathy was applied. Rats were randomized to 4 groups: 1. – intact animals (10 rats, 20 eyes), 2. - rats with NVR on 13th day of observation (10 rats, 20 eyes), 3. - rats with NRV on 45th day of observation (10 rats, 20 eyes), 4. - rats with NRV, intravitreal injected CCBNC on day 13th of observation (10 rats, 20 eyes). Six paraffin-embedded sections prepared in a standard manner and stained with hematoxylin and eosin. To investigate the damage, we evaluated the number of cells in the ganglion cell layer (GCL) and the thicknesses of the retinal layers at a distance between 375 and 625 µm from the optic disc were measured. To determine VEGF and PEDF gene expression levels and RT-PCR was performed. Results: Rat retinas from intact group animals were negative for neovascularization, and showed low levels of fold expression both VEGF and PEDF (0,062 and 0,065 respectively). In the rats from the group 2 morphological patterns of neovascular retinopathy were revealed, it was a dramatic fold expression increase of the VEGF gene and a slight increase in PEDF (2,878 and 0,099, respectively). Rat retinas from group 3 were positive for developed proliferative retinopathy. In this group was the increase of expression of PEDF gene (1,165), despite VEGF gene overweight. In the rat eyes from group 4 at the mid-peripheral retina we observed newly formed vessels at different stages of involution. This animal group revealed a sharp increase in PEDF gene expression and its dominance in comparison with VEGF (1,875 and 0,199 respectively). Conclusion: Regression of neovascular tissue, retinal structure normalization was found after 45 day post single intravitreal injection of cryopreserved cord blood derived nuclear cells.

Purpose: To evaluate the influence of subretinal xenotransplantation of human embryonic stem cell (hESC) derived retinal pigment epithelium (RPE) grown on porous ultrathin carriers in rabbits on retinal preservation. Methods: The hESC-RPE cells were differentiated from two different hESC lines with a previously established xeno-free protocol. The differentiated RPE cells were seeded on human recombinant laminin coated polyethylene terephthalate (PET) inserts with 1.0 µm pores for maturation and characterized by their morphology and polarity, expression of differentiation markers, and phagocytosis activity. Live hESC-RPE monolayer cultured on PET were shipped from Finland to Germany in optimized transportation conditions. TER was measured pre- and post transport. Following vitrectomy, bleb retinal detachments (bRD) were raised with balanced salt solution (BSS) in the eyes of 10 pigmented rabbits. Using custom-made surgical instrumentation, hESC-RPE monolayers on PET were xenotransplanted into the subretinal space (SRS) created by the bRD (N=9). Some of these animals (N=2) received weekly intravitreal injections of the immunosuppressant FK506. As a control, PET without cells was implanted into SRS (N=1). Rabbits were weekly monitored by SD-OCT after implantation. After 4 weeks, the eyes were processed for histology. Results: Human ESC-RPE cells displayed typical RPE characteristics. A logistic protocol for shipment of live hESC-RPE could be established, with comparable pre- and post transport TER and morphology. SD-OCT showed steady positioned implants with an adherent retina over the implant after 4 weeks. A nearly intact xenografted hESC-RPE monolayer could be observed at 4 weeks post implantation to rabbit SRS. SD-OCT and histology showed nearly preserved retinal layers overlying hESC-RPE in some animals (N=5), while in others retinal atrophy appeared at 1 week (N=4). Pilot experiments with intravitreal application of FK506 appeared to alleviate retinal thinning. Control implant with PET alone showed reduced outer retinal thickness without signs of inflammation. Conclusions: These preliminary data suggest for the first time subretinal tolerance of a hESC-RPE monolayer xenotransplant along with improved retinal preservation in rabbits.

PURPOSE: Crystallin β-b2 (crybb2) is known to support the regeneration of retinal ganglion cell (RGC) axons in culture and in vivo and to be expressed in the retinas affected by uveitis. We studied the role of recombinant crybb2 (rcrybb2) on the development of experimental autoimmune uveoretinitis (EAU) in B10.RIII mice. METHODS: EAU was induced in B10.RIII mice by immunization with human interphotoreceptor-retinoid-binding protein peptide 161-180 (hIRBPp161-180). In immunized mice rcrybb2 was intravitreally injected three days prior to immunization, the control mice received PBS as vehicle control, while untreated control mice received no injection. EAU scores were determined histologically on day 21 p.i.. ELISA was performed to measure levels of cytokines produced by splenocytes. The retinas were analyzed by immunofluorescence microscopy, Western blotting, and RT-PCR to detect subsets of different retinal cells, including ganglion cells (betaIII-tubulin, NF-200) and astrocytes (GFAP), as well as apoptosis (Bax, Bcl-2) at day 21 post immunization to monitor neurodegeneration and - regeneration. RESULTS: Crybb2 treated mice immunized with hIRBPp161-180 in complete Freund´s Adjuvants (CFA) with a concomitant injection of pertussis toxin (PTX) showed no significant improvement of EAU with respect to incidence and severity of disease. Markedly reduced expression of selected proteins and associated mRNA levels, including betaIII-tubulin and GFAP were found. Alterations in retinal apoptosis revealed alterations in retinal homeostatis of uveitis-affected retina due to rcrybb2 administration. ELISA analysis of splenocytes from intravitreal treated crybb2 EAU mice showed no significant changes. Splenocytes cultured with crybb2 revealed a slight increase of IL-6, IL-10, IL-17 and TNF-alpha signal. CONCLUSION: The results suggest that intravitreal injection of recombinant crybb2 causes reduction of retinal damage during the EAU, and this effect is independent of local or systemic autoimmune responses.

Introduction: Proliferative Retinopathies, like diabetic retinopathy (DR) and retinopathy of prematurity (ROP), are common sight threatening retinal diseases. Central role in the pathogenesis of both diseases plays the ischemia-induced production of proangiogenic factors. The revascularization/ reperfusion of the ischemic area is central to the resolution of the hypoxia-induced angiogenesis and the ultimate regression of the pathological neovessels. Junctional adhesion molecule-C (JAM-C) is a tight junction molecule expressed in endothelial cells (ECs) that regulates permeability in an Rap-1 dependent manner. In this study we engaged mice with an endothelial specific deletion of JAM-C (EC-JAM-C KO) and their wildtype littermates (EC-JAM-C WT) Methods: Physiological retinal angiogenesis was quantified in postnatal day 6 (p6) in EC-JAM-C KO and WT mice by means of vascular staining in wholemount retinas. The mice were also subjected to the ROP model and at p17 the eyes were enucleated. The pathological neovascularization (endothelial nuclei invading the vitreous cavity) was quantified. Furthermore the ischemic/ avascular area and the tip cell number of the contralateral eye was quantified in retinal wholemount preparations. In vitro, ECs transfected with siRNA against JAM-C or scrambled siRNA were engaged in sprouting assays in the presence or absence of the RAP-1a inhibitor. Results: Athough we found no difference in the physiological or pathological angiogenesiss among endothelial JAM-C sufficient and deficient mice, a significant decrease in the avascular area of EC-JAM-C KO mice that were subjected to the ROP model was noted. This difference was attributed to a higher tip cell number leading the revascularizing vascular plexus in the retinas of EC-JAM-C KO mice as compared to their wildtype littermates. In vitro, the siRNA mediated knockdown of JAM-C led to a significant increase of sprout formation as compared to control transfected cells. This effect was neutralized by a RAP-1 inhibitor. Conclusion: Together, endothelial deletion of JAM-C leads to increased EC sprouting in a RAP-1 dependent manner. In vivo the EC-JAM-C deletion leads to increased tip cell formation and to a more efficient revascularization of the avascular area without affecting the pathological neovascularization. Taken together, our study shows that JAM-C can be an efficient target for the promotion of revascularization in the context of proliferative retinopathies.

Purpose: Last year successful drug delivery via novel lipid DNA-Nanoparticles (NPs) as eye drops to the corneal epithelium was shown. Here we tested distribution and delivery of this class of NPs into the back of the eye to treat retinal diseases. A successful delivery via these NPs would offer chances to reduce the concentration of the drugs or reduce the number of injections needed per year. Methods: Fluorescently labelled NPs were injected either into the vitreous body or into subscleral tissue of ex vivo pig eyes obtained from the local slaughterhouse. After 5, 15, 30, 60 minutes of incubation the eyes were embedded into Tissue Tek and frozen in liquid nitrogen. Subsequently, cryo sections of the eyes were prepared and counterstained with DAPI. Using fluorescence microscopy the NP-diffusion and binding to different structures/tissues (retina, sclera, choroidea and pigment epithelium) was evaluated. Subsequently, the NPs were injected into the vitreous body or the subscleral tissue of living rats. At several time points after injection the same parameters were investigated (30 min, 2, 4, 8, 24 h). Finally, using a fluorophotometer (Fluorotron™) intraocular measurements of NP-concentration and location were performed on rat- and pig eyes in order to analyze diffusion and depot effects. Results: Intravitreal injections into the pig eyes showed good binding to retinal cells, pigment epithelium, choroidal and scleral tissue. Subscleral injections showed similar results with diffusion obviously starting from the sclera and choroid. The in vivo injections into the vitreous body of living rats showed that the NPs bind to most of the tissue in the eye instantly, with residence times lasting up to 24 h. The fluorophotometer results show that the NP stayed inside the eye of living rats longer than the control dye. Conclusion: Our results proof good binding of the NPs to retinal, choroidal and scleral tissue as well as to pigment epithelium with both intravitreal and subscleral injections. Thus, DNA-NPs might be a versatile tool to prolong the residence time of intraocularly injected drugs. Further investigations with medical drugs are necessary to confirm these results.

Fragestellung: In Vorarbeiten konnten wir eine altersabhängige Zunahme von beta-Synuclein (SNCB) innerhalb der Retina und in kortikalen Anteilen des visuellen Systems nachweisen. SNCB dient als physiologischer Gegenspieler des in neurodegenerativen Erkrankungen relevanten alpha-Synucleins (SNCA). Ziel dieser Arbeit war es zu untersuchen, ob SNCB im retinalen als auch im kortikalen Kontext vergleichbare Funktionen sowohl auf neuronale, gliale als auch vaskuläre Zellen in-vitro ausübt. Methodik: Zur Untersuchung von neuronalen und glialen Anteilen der Retina und des primären visuellen Cortex sowie der vaskulären Zellen (Brain Microvascular Endothelial Cells) wurde primäres Gewebe aus der Ratte präpariert und mit rekombinantem SNCB kultiviert. Die Verteilung von SNCB wurde immunhistochemisch evaluiert. Der Einfluss von SNCB auf die Expression von SNCA sowie auf neuronale (beta-III-Tubulin, Neurofilament-200), gliale (GFAP), vaskuläre (Endothelin-1, VEGF) und apoptotische (Bax, Bcl-2) Marker wurde mit immunhistochemischen, proteinbiochemischen (Westernblot) und molekularbiologischen (qRT-PCR) Methoden untersucht. Als Kontrolle dienten nicht-behandelte Zellen. Ergebnis: Es zeigte sich ein konzentrationsabhängiger Effekt von SCNB sowohl auf neuronale als auch vaskuläre Zellen in-vitro. Immunhistochemische Analysen von SCNB demonstrierten Veränderungen auf neuronale (Neurofilament-200), gliale (GFAP), vaskuläre (VEGF) und apoptotische (Bax, Bcl-2) Marker. Darüber hinaus wurden in den Protein- und Genexpressionsstudien signifikante Unterschiede zwischen retinalen und kortikalen Zellen gefunden. Schlussfolgerung: Die hier vorgestellten Ergebnisse deuten auf ungleiche Funktionen von SNCB in unterschiedlichen neuronalen Zielgeweben hin. Weitere Untersuchungen sind notwendig, um genaue Funktionsweisen von SCNB innerhalb unterschiedlicher, neuronaler Gewebe herauszuarbeiten. Die Erkenntnisse lassen jedoch vermuten, dass eine weitere Auseinandersetzung mit altersabhängigen Faktoren im Kontext des neuronalen Zielgewebes zu einem besseren Verständnis neurodegenerativer Erkrankungen beitragen. Gefördert durch Innovative Medizinische Forschung (IMF) der WWU Münster (I-Bö221307)