Postersitzungen, Donnerstag, 29. 9. 2016

 
dt/engl
Posterkabinett 6 13:30 - 14:30 29.09.2016
Postersitzung PDo06
Retina: Grundlagen // Basics
Vorsitzende/r: Nicolas Feltgen (Göttingen), Peter Szurman (Sulzbach)

Referent/in: Nil Celik (Heidelberg)
Fragestellung: Die Pathophysiologie der diabetischen Retinopathie umfasst zelluläre und molekulare Prozesse. Es wird vermutet, dass neben Wachstumsfaktoren auch inflammatorischen Zytokinen an diesen Vorgängen beteiligt sind. Entzündungshemmende Medikamente könnten hier angreifen. Wir untersuchten die Wirkung der oralen Verabreichung einer Kombination aus Zink und Acetylsalicylsäure [Zn(ASA)2], auf Veränderungen der Genexpression in der Netzhaut in Zucker diabetic fatty (ZDF) Ratten, die einen Typ-2-Diabetes entwickeln. Methodik: Nicht-diabetische Kontrollraten (ZL) und obese ZDF Ratten wurden für 24 Tage oral mit Placebo oder Zn(ASA)2 vorbehandelt. Im Alter von 29-30 Wochen wurde retinale RNA isoliert. Mittels reverser Tranksriptase wurde die cDNA synthetisiert um die Expression 84 ausgewählter inflammatorischer Gene mithilfe der PCR über „micro fluid cards“ bestimmt. Ergebnisse: Der Plasmaglukosespiegel war bei Ratten behandelt mit Zn(ASA)2 signifikant erniedrigt (39 ± 4 mM vs 49 ± 3 mM, p < 0,05), während die Serum -Insulin-Konzentration unter den Gruppen ähnlich war. Die retinale Expression einiger inflammatorischer Gene (BMP2, CCL2, CCL20, CCL3, CCL4, CCL7, CCR1, CCR3, CXCL1, CXCL6, CXCL9, FASLG, IL13, IL15, IL17A, IL17B, IL1B, IL33, IL4, TNF, VEGF-A und VFRSF11B) war mindestens zweifach höher in ZDF im Vergleich zu den ZL Ratten. Im Vergleich zur Placebogruppe zeigte sich in der behandelten Gruppe mit Zn(ASA)2 eine signifikante Herabregulation bestimmter inflammatorischer Gene (CCL2, CCL20, CCL3, CXCL6, IL13, IL17A und VEGF-A) bei obese ZDF-Ratten. Schlussfolgerung: Inflammatorische Gene waren in der Retina fettleibiger ZDF-Ratten im Vergleich zu den ZL Tieren signifikant hochreguliert. Die orale Verabreichung von Zn(ASA)2, die den Blutzuckerspiegel senkt, führt zu reverseierten Veränderungen in der Genexpression bei Ratten mit diabetischer Retinopathie. Basierend auf diesen Ergebnissen sind weitere Untersuchungen zu Pathway-Analysen notwendig um bei Typ-2 induzierter Retinopathie auf molekularer Ebene genauere Erkenntnisse zu gewinnen. Gefördert durch die Ernst und Berta Grimmke Stiftung
Referent/in: Ivanka Dacheva (Heidelberg)
To analyze the levels of lysophosphatidic acids (LPAs) and autotaxin (ATX) in undiluted vitreous of untreated patients with retinal vein occlusion (RVO). Sixty-four vitreous samples (40 RVO, 24 controls with idiopathic floaters) were analyzed in this retrospective case series using LC/MS for LPAs 16:0, 18:0, 18:1, 20:4, and ELISA kits or Luminex technology for ATX, angiopoetin-1 (ANG-1), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), pigment epithelium-derived factor (PEDF), vascular endothelial growth factor (VEGF). LPA and ATX levels were correlated with the visual acuity, central macular thickness (CMT), average retinal thickness (AvT), vitreal cytokine levels and with each other. Levels of every LPA species tested and ATX were significantly increased in the vitreous fluid from all patients with RVO (total LPAs: 968.0±842.3 nM; ATX: 2.5±1.02 nM) compared to controls (total LPAs: 225.2±292.8 nM, p< .0001; ATX: 1.9±1.00 nM, p=.005). There were strong positive correlations between the vitreal levels of IL-6, IL-8, MCP-1, VEGF and LPAs. Levels of LPAs and ATX were positively correlated with proinflammatory cytokines and VEGF and might thus play an important role in the development of macular edema secondary to RVO.
Referent/in: Franziska Ludwig (Freiburg)
Purpose: Retinal microglia (MG) have been implicated in the development of the retinal vasculature and the formation of pathological retinal neovascularization. The interferon regulatory factor 8 (IRF8) is a central transcription factor for myeloid cell maturation and differentiation. The aim of this study was to determine the role of Irf8 for MG cell distribution during retinal development and in the process of retinal neovascularization by characterizing Irf8-deficient knock-out mice. Methods: Irf8-Venus reporter mice were characterized by immunohistochemistry and flow-cytometry during development and in the oxygen-induced retinopathy (OIR) mouse model. MG cell distribution was analyzed in Irf8-/-/Cx3cr1+/GFP (Irf8 KO) and age-matched Cx3cr1+/GFP control mice (Irf8 WT) at postnatal day (P) 3, 5, 7, 12, 17, 24 and 31. Finally, Irf8 KO and Irf8 WT mice were assessed in the OIR mouse model. The area of ischemia and retinal neovascularization was determined by immunohistochemistry on retinal flatmounts at P12 and P17. Results: Using Irf8-Venus reporter mice, we found that all MG express Irf8 during retinal development and that Irf8 expressing MG accumulate at sites of retinal neovascularization in the OIR model. Irf8 KO mice exhibited a significantly reduced number of MG during retinal development. In the OIR mouse model, Irf8 KO mice developed a larger ischemic area at P12 and P17 compared to Irf8 WT controls, whereas the area of retinal neovascularization was similar in both groups. Conclusion: Irf8 is expressed in retinal microglial cells under physiological and pathological conditions and is vital for MG cell distribution during development. In the OIR mouse model mice deficient for Irf8 exhibit a larger avascular zone indicating Irf8 as a critical factor for revascularization.
Referent/in: Clemens Lange (Freiburg)
Background: Myeloid cells, such as resident microglia (MG) and circulating monocytes (MO), are cells of the innate immune system which are implicated in neurovascular development, retinal tissue homeostasis, and the formation of choroidal neovascularization (CNV). The Interferon Regulatory Factor 8 (Irf8) is a central transcription factor for myeloid cell maturation, differentiation and lineage commitment. The aim of this study was to determine the expression pattern of Irf8 in the retina and to assess the role of Irf8 for retinal neurovascular homeostasis and formation of CNV. Methods: To determine the expression profile of Irf8 in the blood and retina, adult Irf8 Venus reporter mice were studied by flowcytometry and immunohistochemistry in the steady state and in a model for laser-induced CNV. Next, adult Irf8 knockout mice (Irf8 KO) and age-matched controls (Irf8 WT) were analyzed for MG cell numbers, density and morphology by flowcytometry and immunohistochemistry. The retinal vasculature, structure and function were assessed by immunohistochemistry, optical coherence tomography (OCT) and electroretinography (ERG) in Irf8 KO and Irf8 WT, respectively. Finally, the role of Irf8 for choroidal neovascularisation was analysed using the laser-induced CNV model. Results: Irf8 reporter mice revealed that Irf8 is exclusively expressed in retinal bipolar cells and MG in the retina and in monocytes of the blood. Irf8 KO mice exhibited a severely altered MG morphology and reduced MG cell numbers compared to Irf8 WT. In the blood, Irf8 KO demonstrated loss of circulating monocytes and increased numbers of neutrophiles. The retinal vasculature, structure and neuroretinal function was similar in adult Irf8 KO and WT animals. In the laser-induced CNV model, however, Irf8-deficient mice exhibited reduced myeloid cell recruitment to sites of laser injury and increased size of CNV compared to WT controls. Conclusion: Our study demonstrates that Irf8 is vital for MG homeostasis and function but not for retinal neurovascular development. Our findings indicate that Irf8 is a critical factor for transforming microglia into a reactive phenotype thereby suppressing retinal inflammation and the formation of CNV.
Referent/in: Claudia Brockmann (Berlin)
Hintergrund: Die choroidale Neovaskularisation (CNV) stellt die fundamentale Pathologie der altersbedingten Makuladegeneration dar. Im experimentellen Ansatz hierfür ist das Mausmodell der Laser-induzierten CNV anerkannt. Um den Einfluss von Makrophagen und Mikrogliazellen auf die CNV zu untersuchen, induzierten wir erstmals im transgenesen Mausmodell CD11b-HSVTK (Cluster of differentiation CD11b, herpes simplex virus thymidine kinase, B6,D2-Tg (CD11b-HSVTK)620Zbz) die CNV. Methoden: Die Verteilung retinaler CD11b-positiver Zellen wurde nach intravitrealer Injektion von Ganciclovir (GCV, Depletionsgruppe) und Natriumchlorid (NaCl, Kontrollgruppe) in transgenen (TK+) und nicht-transgenen Geschwistertieren (TK-) zu verschiedenen Zeitpunkten analysiert. Post mortem wurde die Auswirkung der Reduktion CD11b-positiver Zellen auf die Ausprägung der CNV im retinalen Flachpräparat untersucht. Ergebnisse: Drei Tage nach GCV-Injektion verringerte sich die Zahl der CD11b-positiven Zellen um rund 20%, nach 7 Tagen um 40% in TK+ Tieren. Alleinige Injektionen von NaCl führten zu allen Zeitpunkten zum Anstieg der CD11b-Zellzahl gegenüber Augen ohne intravitreale Injektionen. Es zeigten sich keinen signifikanten Unterschiede in der Größe der CNV nach maximaler lokaler Depletion CD11b-positver Zellen in TK+ Tieren. Schlussfolgerungen: Im CD11b-HSVTK Mausmodell ist eine lokale Depletion retinaler CD11b-positiver Zellen möglich. Eine Reduktion der CNV wurde dadurch nicht erreicht. CD11b-positive Makrophagen und Mikrogliazellen allein scheinen einen begrenzten Einfluss auf die Ausprägung der CNV im Mausmodell zu haben.
Referent/in: Yvonne Nowosielski (Innsbruck)
Background: Wet age-related macular degeneration (AMD) is characterised by the formation of choroidal neovascularisations (CNVs) which sprout from the choroid into the subretinal space causing macula edema and/or bleeding resulting in central vision loss. Vascular endothelial growth factor is known to be the major involved growth factor in choroidal angiogenesis. In this study we aimed to evaluate firstly whether the neuropeptides substance P (SP) and neuropeptide Y (NPY), which act in a proangiogenic manner with a potency similar to that of VEGF, contribute to the pathogenesis of CNVs. Secondly we evaluated two different techniques for CNV volume measurement: a rather new in vivo measurement technique using spectral-domain optical coherence tomography (SD-OCT) and the commonly used ex vivo flatmount technique. Methods: Volumes of laser- induced CNVs were obtained on day four after laser application. Each mouse eye was then analysed in vivo by SD-OCT. For the ex vivo method mice were sacrificed, eyes enucleated, flatmounted and stained for von Willebrand factor. Volumes were obtained by analysing z-stacks taken by a laser scanning confocal microscope with Imaris software. Seven C57bl6/N wildtype mice were compared to five Y2- (NPY2-receptor) and four Nk1- (SP-receptor) knockout mice of the same background. Differences in the CNV volume of single spots between wildtype and knockout mice were analysed with Mann-Whitney U test (MWU). Five wildtype, five Y2-knockout and four Nk1-knockout mice with C57bl6/N background were used for comparison of the two methods. Pearson correlation was performed on mean volumes of CNV spots per mouse. Results: Comparing CNV spots in wildtype (n=14) with Y2-knockout (n=22) and Nk1-knockout mice (n=20) no difference in mean OCT volume could be detected (p=0.360, p=0.204, respectively, MWU). No difference in mean volume could be detected either using choroidal flatmounts for volume measurements (p=0.309, p=0.133, respectively, MWU). In comparison the two methods used to obtain CNV volumes showed a significant correlation (R=0.543, p=0.045, n=14 mice). Conclusion: Despite the fact that there was no significant difference between CNV volume comparing knockout to wildtype mice, knockout mice generally still showed reduced volumes. Therefore we decided to add further analyses including a higher number of mice. OCT is useful as a non-invasive tool for the in vivo measurement of laser-induced CNVs.
Referent/in: Larissa Lahme (Münster)
Fragestellung: Die Anti-VEGF-Medikation hat sich als first-line-Therapie bei verschiedenen neovaskulären Augenkrankheiten etabliert. Allerdings gibt es Patienten, die nicht optimal auf diese Therapie ansprechen. Dies bedingt die Notwendigkeit nach neuen Ansätzen und Zielmolekülen bei der Therapie von neovaskulären Erkrankungen zu suchen. Das Ziel der vorgestellten Studie ist deshalb die Identifizierung von Signalwegen abseits von VEGF, die ebenfalls bei dem Wachstum und der Reifung von neovaskulären Blutgefäßen eine Rolle spielen könnten. Methodik: Es wurde das Modell der Laser-induzierten choroidalen Neovaskularisation an C57BL/6-Mäusen angewendet. Um den zeitlichen Verlauf darzustellen wurden die Augen ein, zwei, drei und vier Wochen nach der Laserbehandlung entnommen. Die Expression der Gene verschiedener Wachstumsfaktoren, Zytokine und Rezeptoren wurde mittels der quantitativen PCR analysiert. Ergebnisse: Während die mRNA für RPE65 in der ersten Woche nach der Laserbehandlung deutlich verringert war und bis zur vierten Woche wieder anstieg ohne jedoch das Ausgangniveau zu erreichen, zeigte sich bei CD31 (Endothelzellen), SMA (Perizyten) und S100A4 (Fibroblasten) nach einer zum Teil deutlichen Steigerung innerhalb der ersten Woche wieder eine Normalisierung auf das Ausgangsniveau. Die Genexpression der Wachstumsfaktoren PDGF-β, TGF-1-β und FGF-2 zeigte eine Woche nach der Laserbehandlung einen deutlichen Anstieg und sank dann auf die Ausgangswerte herab. Im Gegensatz dazu blieb die Expression von FGF-1, VEGF und des PDGF-β-Rezeptors im Laufe der vier Wochen nahezu unverändert. Die mRNA der Zytokine MCP-1, IL-1-β und IL-6 war ebenfalls eine Woche nach der Laserbehandlung erhöht und sank anschließend wieder ab. Die Expression von MCP-2 stieg gleichmäßig über den Beobachtungszeitraum von vier Wochen leicht an. Schlussfolgerungen: Die stärksten Änderungen der Genexpression zeigten sich eine Woche nach der Laserbehandlung, wohingegen nach vier Wochen kaum noch Unterschiede zum unbehandelten Auge auszumachen waren, mit Ausnahme des Chemokins MCP-2. Auffällig war auch die starke vorübergehende Hochregulation von PDGF-β, TGF-1-β und FGF-2.
Referent/in: Jakob Siedlecki (München)
Fragestellung: Alle derzeit für die neovaskuläre AMD zugelassenen Therapeutika bieten ausschließlich eine anti-VEGF Monotherapie. Unberücksichtigt bleibt dabei die durch den platelet derived growth factor (PDGF) vermittelte Stabilisierung choroidaler Neovaskularisationen durch Perizyten, die krankhafte Gefäße von außen umfassen und eine partielle anti-VEGF Resistenz vermitteln, die zur hohen Therapiefrequenz des derzeitigen therapeutischen Goldstandards beiträgt. In der folgenden Arbeit wird untersucht, ob eine duale Inhibition von VEGF- und PDGF-Signalwegen durch den Tyrosinkinaseinhibitor Axitinib in einem in vitro-Modell der neovaskulären AMD angiogene Zellfunktionen humaner Endothelzellen und Perizyten inhibieren kann. Methodik: Humane Endothelzellen (HUVEC) und Perizyten (hPC-PL) wurden mit Axitinib-Konzentrationen von 1 ng/ml bis 10 µg/ml behandelt. Mittels modifiziertem MTT-assay wurden Toxizität und Proliferation gemessen, während zelluläre Migration in einem Boyden Chamber-Assay unter Stimulation mit VEGF und PDGF beobachtet wurde. Der Einfluss von VEGF und Axitinib auf die Ausbildung kapillärer Strukturen wurde auf Wachstumsfaktor-reduzierten und regulären Cultrex-Gelen untersucht, die eine an pro-angiogenen Wachstumsfaktoren reiche Basalmembran bilden. Ergebnis: Axitinib ermöglichte eine signifikante, dosisabhängige Reduktion zellulärer Proliferation von Endothelzellen und Perizyten. VEGF und PDGF führten zu einer signifikanten Induktion zellulärer Migration, während die simultane Behandlung mit Axitinib diesen Effekt wieder rückgängig machte. VEGF induzierte eine signifikante Verlängerung kapillärer Strukturen auf Wachstumsfaktor-reduzierten Cultrex-Gelen; dieser Effekt wurde durch Axitinib signifikant reduziert. Zusätzlich führte Axitinib bei maximaler pro-angiogener Stimulation auf regulärem Cultrex-Gel zu einer Abnahme der Gesamtlast perivaskulären Gewebes, das die entstehenden Kapillaren begleitet. Schlussfolgerung: In vitro führt Axitinib über die simultane Modulation von VEGF- und PDGF-Signalwegen zu einer signifikanten Reduktion angiogener Prozesse von humanen Endothelzellen und Perizyten. Weitere Studien müssen klären, ob diese duale Inhibition auch in vivo eine länger wirksame und effektivere Therapie der neovaskulären AMD ermöglicht.
Referent/in: Ekaterina Rzhavina (Moscow)
Purpose: Various pathways are involved in cell death after the ischemia. Arachidonic acid cascade is believed to be one of the components of the ischemic injury. Consequently, block of cyclooxygenase 1 or 2 (COX) or phospholipase A2 could have a neuroprotective effects on ischemic retina. Therefore, the purpose of this study was to access the role of arachidonic acid cascade in retinal ischemic damage. Methods: Eye ischemia was modelled by irreversible bilateral internal carotid arteries occlusion in Wistar male rats. In 15 min after artery ligation lornoxicam (0.008 mg/μl, 2 μl) or triamcinolone (0.04 mg/μl, 2 μl) or saline (2 μl) was injected intravitreally. Maintenance therapy of respective therapeutics was performed during first 2 days parenterally. Fundus examination was carried out on 1, 3, 7, 14, 28, 56 days of the experiment and in 6 month. Enucleation was performed at the same time points. Retinal remodeling and thickness of retinal layers were examined. COX 1, 2 expression was evaluated by immunohistochemical method. Results: Ophthalmoscopic examination shown venous dilation and stasis and further vessels degradation.Triamcinolone maintained capillary perfusion but had short-term effect and increased risk of edema. Fundus imaging in lornoxicam group shown the longest vessels degradation and no retinal edema. Retinal thickness decreased during whole experiment in all groups. Triamcinolone and lornoxicam retarded retina thinning; moreover, lornoxicam prevented undulating remodeling retina. COX 2 expression increased in the internal nuclear (INL) and ganglion cell layers (GCL) after ischemia and by the 56th day of the experiment slowly decreased. COX 1 was present in both plexiform (IPL, OPL) and GCL. After ischemia injury, COX-1 expression slowly diminished in the GCL. Triamcinolone reduced COX-1 expression in the IPL and did not influence on COX-2 in a long-term. In lornoxocam group COX-1 expression increased in the IPL and then slowly normalized and COX-2 immunoreactivity decreased. Conclusions: Bilateral internal carotid arteries occlusion caused slowly reduction of retinal thickness, worsening of fundus circulation and upregulation of COX-2. Triamcinolone had short-term effects on retina remodeling. Intravitreal lornoxicam injections had significant neuroprotective effects on ischemic retina. This study was funded by a RFBR grant № 14-04-01318-a.
Referent/in: Cara Rodust (Bochum)
Purpose: N-Methyl-D-Aspartate (NMDA) leads to a significant loss of ganglion cells in retina and destruction of optic nerves. But the apoptotic pathways in the NMDA animal model are barely known. This project is set to detect the connection between cell death, microglia cells and proapoptotic factors in a NMDA induced damage in retina and optic nerve. Methods: Rats were intraocularly injected with a NMDA concentration series (0 (PBS), 20, 40, 80 nmol) and retinae and optic nerves (ON) were examined 14 days after injection (n=5-6/group). Ganglion cell loss was shown with Brn-3a Western blot analysis. Destruction of ON was detected with SMI-32. Iba1 and ED1 staining represented immigration and activation of microglia. Complement system involvement was shown with C3 and MAC staining. To outline the apoptosis mechanisms in this model FasL/FasR staining was used. Statistical analysis was performed with ANOVA followed by Tukey post-hoc test. Results: All results compare NMDA groups to PBS injection. 40 and 80 nmol NMDA injection led to ON destruction (p=0.0009). FasL/FasR colocalisation in ON was increased with 80 nmol NMDA (p=0.01), while in retina no elevated apoptosis rates were detectable in any NMDA group (p>0.5). Microglia immigrated into retina after NMDA injection (20 nmol p=0.01, 40 / 80 nmol p< 0.0001) and were activated with high NMDA concentrations (40 nmol p=0.0004, 80 nmol p=0.0006). Activated microglia were also found in ON with 40 nmol NMDA (Iba1 / ED1 p< 0.0001) and 80 nmol NMDA (Iba1 p=0.005; ED1 p=0.002). Injection of 80 nmol NMDA increased the level of the complement factors C3 (p=0.0001) and MAC (p=0.007) in the retina, while no alterations were found in optic nerves (p>0.5). Conclusion: NMDA led to destruction of tissue in retina and ON. Apoptotic processes in the ON were detected 14 days after injection, while in retina, the primary place of cell death, no apoptosis was seen at this late timepoint. Activation of microglia remained on high levels. Probably oxidative stress and loss of neurons made this long lasting activation possible, even after the apoptosis rate decreased. Active microglia can secret complement factors, which were elevated in retina and can again induce apoptosis. The interaction of apoptosis, microglia activation and complement factor elevation plays a major role in this NMDA-induced degeneration model.
Referent/in: Sandra Kuehn (Bochum)
Purpose: Cobalt as part of the vitamin B12 is important for the neuronal integrity. Yet, it is known that high quantities of cobalt induce cytotoxic and neurotoxic mechanisms, which could lead to hypoxia. Therefore, we tested the degenerative effect of cobalt chloride (CoCl2) on neurons and microglia in a retina organ culture model. The aim is to establish an alternative retina degeneration model to reduce the number of animal experiments. Methods: Organotypic cultures of porcine retina were cultivated and treated with different concentrations of CoCl2 (0 (control), 100, 300 and 500 µM) for 48 h from day 1 up to day 3. At day 8, retinas were cryo-conserved for histological (n=8/group), Western blot (n=4-5/group) and qrt-PCR (n=5/group) analysis. Retinal ganglion cells (RGCs, Brn-3a), amacrine cells (Calretinin) and bipolar cells (PKCα) were counted. Additionally, microglia (Iba1) in an active state (CD16/32) were analyzed. Groups were compared with one-way ANOVA with post-hoc Dunnett’s test. Results: CoCl2-concentrations of 300 and 500 µM, led to a decrease of the Brn-3a+ ganglion cells (300 µM: p=0.002; 500 µM: p< 0.001), Calretinin+ amacrine cells (300 µM: p=0.002; 500 µM p=0.001) and PKCα+ bipolar cells (300 µM: p=0.007; 500 µM: p=0.001). In contrast, 100 µM CoCl2 had no effect on the neurons of the porcine retina (Brn-3a, Calretinin, PKCα: p>0.05). In addition, all three CoCl2 concentrations reduced the microglia population (100 µM p=0.07; 300 µM: p< 0.001; 500 µM: p< 0.001). The same applies for activated microglia (CD16/32; 100 µM: p=0.001; 300 µM: p< 0.001; 500 µM: p=0.02) Conclusions: CoCl2 induced a strong degeneration of the porcine retina, starting at 300 µM. Especially the neurons of the inner retina layers were affected. However, the degenerative effect of CoCl2 was not restricted to neurons, also the microglia underwent a death mechanism. These damaging effects on microglia were surprising, since CoCl2 causes hypoxia and a pro-inflammatory environment. Yet, high concentrations of CoCl2 seem to be generally toxic for these cells. Compared to a retinal ischemia animal model, similar degenerative mechanisms in regard to neuronal cell loss were observed in this novel organ culture model. In conclusion, a very promising alternative model for retinal degeneration could be established.