Poster Sessions Saturday

Purpose: Aqueous humor of patients with primary open angle glaucoma (POAG) contains elevated amounts of transforming growth factor (TGF)–β2 contributing to an increased outflow resistance by an accumulation of extracellular matrix (ECM) material in the trabecular meshwork (TM). In vitro studies of cultured TM cells treated with TGF-β2 elucidated two mechanism leading to the ECM alterations. TGF-β2 lead to an increased ECM synthesis mediated through the connective tissue growth factor (CTGF) and by inhibiting the activation of matrix metalloproteinases (MMP) activiation via serine proteinase inhibitor. The serine protease inhibitors beside the plasimogen activator inhibitor-1 (PAI-1) are poorly investigated in the TM. We have identified the tissue factor pathway inhibitor-2 (TFPI-2) by microarray analysis as a potential new candidate of the serine protease inhibitors contributing to fibrogenic effects of TGF-β2 and CTGF in the TM. Methods: Cultured TM cells from 4 human donors were treated with 1ng/ml TGF-β2 and with 50ng/ml CTGF and 100ng/ml CTGF for various time points. The expression of TFPI-2 was quantified by real-time RT-PCR, Western blot analysis and by immunohistochemistry. The in vivo expression of TFPI-2 in the outflow tissue of a murine glaucoma model (βb-1CTGF) induced by a transgenic eye specific overexpression of CTGF was analyzed in comparison to wild-type animals at the age of 2 and 3 month. Results: The in vitro treatments with TGF- β2 and CTGF lead to an induction of the TFPI-2 expression and synthesis in human TM cells. The in vivo analysis of the TFPI-2 expression rate in the βb-1CTGF mice in comparison to the wild-type littermates could confirm the in vitro data. The βb-1CTGF mice have elevated level of TFPI-2 at both investigated time points. Conclusion: TGF-β2 and CTGF induced upregulation of TFPI-2 could contribute to the ECM accumulation by an inhibition of MMP activity. An reduced MMP activatiy might be responsible for the observed elevation of the IOP in the murine glaucoma model, pointing into the direction that TFPI-2 is a new candidate molecule leading to an increased outflow resistance in the TM of POAG patients.

Background: Several studies demonstrate an autoimmune component for the disease glaucoma, showing not only up-regulated autoantibodies, but also down-regulated antibodies. One of these antibodies (ab) is targeted against ɤ - synuclein and is down-regulated in glaucoma patients. In previous studies we detected a neuroprotective effect of ɤ- synuclein antibodies on stressed RGC5 cells. With the following study we wanted to validate the neuroprotective effect of the antibody on retinal ganglion cells (rgc) in an organ culture. Materials and methods: The retina and retinal pigment epithelium (RPE) was isolated from porcine eyes derived from 3-6 month old pigs. The RPE was placed on nitrocellulose and the retina was placed on the RPE, rgc facing upwards. Using Transwell inserts, the retinae were incubated with medium containing 10 % FBS either with, or without additional 0.5 µg/ml ɤ- synuclein ab (n= 4). After 24 h the retinae were fixed in paraffin and stained for BRN3a, with a tunel assay and DAPI. Retinal ganglion cell counts were performed for 6 slices per retina. Also proteomic measurements of the retina incubated with the antibody as well as the control retina were performed. Results: We were able to detect an increase in RGC/mm of 41 % in the retinal organ culture incubated with 0.5 µg/ml ɤ- synuclein ab (p < 0.01; standard deviation 2.3). Tunel staining was equal in both groups. The proteomics analysis found 406 proteins in total and showed a significant increase of glutamine synthetase expression of 4.5 fold in the retina incubated with ɤ- synuclein ab as well as significantly increased CDC 42 (9.5 fold) and decreased reticulon 4 (-2.8 fold), both involved in endoplasmic reticulum stress. Conclusions: In this study we were able to confirm the neuroprotective effect of the ɤ- synuclein ab on retinal ganglion cells. Increased glutamine synthetase expression levels demonstrate that Mueller cells, which are important for the homeostasis of the retina, are also affected by the antibodies and are involved in the protective mechanisms. Additionally, endoplasmic reticulum stress proteins such as CDC 42 and reticulon 4 seem to play a role in the protective mechanism of the antibody.

Purpose: When the RGC-5 cell line was established it was positive for ganglion cell markers and showed features of ganglion cells. However, different authors reported about dedifferentiation of the cell line. Even more, there is strong evidence that RGC-5 cells might be contaminated with the 661W photoreceptor cell line. Nevertheless, last year we presented, that there are differences between 661W and RGC-5 cells. The aim of this study was to reveal if RGC-5 cells still show features of ganglion or neuronal cells under conditions of differentiation with Trichostatin A (TSA). Methods: RGC-5 cells were treated for 24, 48, 72, 96 & 120 h with 40, 150, 500 and 2000 nM of TSA according to two published protocols (Schwechter et al. 2007 & Wood et al. 2010) to cause redifferentiation. Cell morphology, death, amount and viability were analyzed. Caspase 3/7-Assays were performed to detect apoptosis. The expression of neuronal markers such as MAP-2, tau, neurofliament and βIII-tubulin was investigated via immunofluorescence and Western-Blot. Relative expression of the ganglion cell marker Thy-1 was detected via qRT-PCR. Results: RGC-5 treated with 500 or 2000 nM of TSA appeared with various neuritic outgrows no matter which protocol was applied. Immunostaining and Western-Blot showed increased expression of the investigated neuronal markers after long-term incubation with 500 nM TSA. The relative expression of Thy-1 mRNA increased more than 2-fold in RGC-5 incubated with 500 nM according to Wood’s protocol. With regard to apoptosis and cell death, these were mainly detected during the first days of incubation while long term treatment (96 to 120 h) with higher TSA concentrations did not cause significant high cell death or apoptosis. In accordance, cell viability of TSA treated cells reached a peak after 120 h of incubation with 500 nM TSA using both protocols. Conclusion: Long-term TSA treatment with 500 nM according to Wood’s protocol caused increased Thy-1 mRNA and the highest observed β-III-tubulin expression as well as a stop of proliferation while cell amount remained relatively constant and viability of cells increased. Therefore we see the highest chance of re-differentiation in treating RGC-5 cells with 500 nM over 120 h according to Wood’s protocol. Nevertheless, it is only a slight movement towards a neuronal and perhaps ganglion cell-like phenotype and using RGC-5 cells in context of ganglion cell research cannot be recommended at the current stage.

Goal: To determine whether change of the retinal arterio-venous oxygen saturation (A-V) difference secondary to flicker light stimulation is associated with the retinal nerve fiber layer (RNFL) thickness in glaucoma patients. Methods: In 41 patients (64.1 ± 12.9 years), suffering from primary open angle glaucoma (POAG), RNFL thickness was measured by frequency domain OCT (Cirrus, Carl Zeiss Meditec AG) and retinal vessel oxygen saturation (SO2) was measured by dual wavelength oximetry (Retinal Vessel Analyser, Imedos Systems UG) in the superior (S), nasal (N), and inferior (I) retinal quadrant before and during a 12.5 Hz luminance flicker lasting for 90 seconds. Results: The flicker stimulus significantly (p< 0.05) increased the venous SO2 by 2.9%, 2.5%, and 2.1% (S, N, and I respectively) and decreased the A-V difference by 3.3%, 2.4%, and 2.7%. There was no significant change of the arterial saturation. Furthermore, neither the venous SO2 nor the A-V difference correlated with the RNFL thickness in the single quadrants. S and I quadrants with RNFL thickness below the 1^st percentile of the normative database showed no different flicker induced changes of the venous SO2 or the A-V difference than quadrants above 1^st percentile. Conclusions: In contrast to the change of vessel diameters under flicker by neuro-vascular coupling, which was found to depend on RFNL thickness in an earlier study, the A-V- difference in SO2 did not depend on the RNFL. This indicates that oxygen supply and consumption in the glaucomatous retina is not critically affected by RNFL thickness.

Purpose: Early molecular events of abnormally elevated intraocular pressure (IOP) remain obscure. Understanding of the pathophysiological mechanisms involved in glaucomatous damage remains one of the major targets of experimental efforts. It is still a matter of debate whether neurons or capillary cells are primarily damaged by elevated IOP. We developed new techniques to scrutinize the molecular mechanisms involved and the cells primarily affected in glaucoma: The neurons or the capillaries? Methods: In vivo, elevation of IOP occurred experimentally after cauterization of episcleral veins. In vitro, dissociated retinal or brain cells were exposed to controlled elevated pressure (30 and 60 mmHg) within a newly fabricated incubation chamber that allowed simulating controllable high IOP over days in culture. In vitro, organotypic retinal explants were cultured under controlled IOP-elevation within the high pressure chamber (30 and 60 mmHg) over days. Immunohistochemistry, Western blotting and qRT-PCR was performed to analyze several molecular markers including ß-III tubulin, VEGF, endothelin-1, GFAP, GAP-43, Iba-1, vimentin, alpha-SMA, PDGFR-beta, desmin and von Willebrand factor VIII. Results: ß-III Tubulin, which is normally a selective marker for developing neurons and only outlines ganglion cells in the retina, is abnormally expressed in both endothelial capillary cells and pericytes after elevation of IOP both in vivo and in vitro. The capillary cell responses preceded the neuronal responses. Neuronal expression on ß-III tubulin remained constant in RGCs. In retinal explants cultured at high pressure in vitro, ß-III tubulin was not upregulated in capillaries indicating that the regulation of its expression needs the intracapillary perfusion as occurring in vivo and after cell dissociation. Double staining with various markers and ß-III-tubulin revealed that both pericytes (alpha-SMA, desmn, NG-2 and PDGFR-ß-positive) and endothelial cells (ET-1, vW-VIII-positive) were affected. Conclusion: Retinal capillaries respond sensitively to abnormal IOP-elevation by early expression of ß-III tubulin preceding neuronal responses. The capillary responses may influence secondary neuronal responses which culminate in death of ganglion cells and blindness as occurs in clinical glaucoma.

Purpose: Previous studies showed that intraocular NMDA injection leads to retinal ganglion cell (RGC) death. This time, the focus is set on the structural alteration and cell death in the optic nerve. Furthermore, we examined the involvement of microglia in the cell death cascade in this model. Methods: One eye per rat was intraocularly injected with NMDA in a concentration series from 0 (PBS), 20, 40 to 80 nmol/2µl (n=5-6/group). The other eye was used as untreated control (untreated). 14 days after injection, apoptotic cells in retina and optic nerve were counted (cleaved caspase 3) related to the absolute number of RGC (Brn-3a) and nuclei (DAPI). Structural effects of NMDA in the optic nerve were shown with SMI-32 staining. Microglia immigration and activation was represented by Iba1 and ED1 positive cells. The results were statistically analyzed with one-way ANOVA, Tukey post-hoc test. Results: All results compare NMDA groups to the PBS group. Both the RGC loss (Brn-3a: 40 nmol p=0.008; 80 nmol p< 0.0001) and structural disorganization in optic nerve was detectable in groups of 40 and 80 nmol NMDA (SMI-32: 40/80 nmol p=0.0009). Apoptotic cells in retina showed no significant differences in NMDA treated groups (cl. caspase 3/Brn-3a ratio: p>0.05). In the optic nerve the number of apoptotic neurons was increased in the 40 and 80 nmol NMDA group(cl. caspase 3/DAPI: 40 nmol p=0.0002; 80 nmol p=0.002). Microglia immigrated in retina (Iba1: 20 nmol p=0.01; 40/80 nmol p< 0.0001) and optic nerve (Iba1: 40 nmol p< 0.0001, 80 nmol p=0.005) in an active state after 40 and 80 nmol NDMA injection (ED1: retina 40 nmol p= 0.0004; 80 nmol p=0.0006, optic nerve 40 nmol p< 0.0001; 80 nmol p=0.002). Conclusion: 14 days after NMDA injection a destruction of optic nerve structure could be observed dependent on the concentration. Corresponding to previous studies the number of RGCs was reduced in groups of 40 and 80 nmol NMDA. NMDA is a trigger to cell death in retina and optic nerve, but optic nerve degenerates later than retinal structures. Then, a higher number of apoptotic cells was only detectable in the optic nerve and no longer in the retina. The reason probably is the late point of examination, because the initial damage took place in the retina, the primary location of injection. This suggests that the intravitreal injection of NMDA causes a secondary apoptosis cascade in the optic nerve which was set later than the retinal apoptosis.

Introduction: Glaucoma represents a group of eye disorders partly related to raised IOP leading to progressive optic nerve damage resulting in impaired vision and possibly blindness. To date, rabbit glaucoma models described in literature are difficult to reproduce, and are often associated with high levels of discomfort and pain for the treated animals. Here, we describe the adaption of oculopressure tonometry (OPT), an existing method to quantify the outflow of aqueous humor in humans, to generate temporary IOP elevation representing a transient in vivo rabbit glaucoma model. Methods: Adaption of OPT to the animal eye was done using New Zealand White rabbits. Measurements of the intraocular pressure (IOP) were carried out with an Icare^® rebound tonometer. To increase the IOP in anesthetized rabbits a suction-cup oculopressor (SCOP) was adjusted to 40 mmHg and custom made suction-cups with different diameters (10, 11 and 12 mm) were placed between limbus and temporal canthus. IOP was measured before and every minute during OPT, followed by a final measurement after OPT termination. Results: OPT was successfully applied to increase the IOP in New Zealand White rabbits. The suction cup with 11 mm in diameter was most suitable in our laboratory settings and yielded the best match with regard to the suction-cup/eyeball diameter ratio used in human measurements. With this suction cup an increase of IOP to 45.8 ± 14.2 mmHg for the right eye (OD) and 50.0 ± 12.3 mmHg for the left eye (OS) could be obtained at the beginning of measurements (n = 9, respectively). These elevated IOP values decreased over a time period of nine minutes to 29.9 ± 13.3 mmHg and 31.0 ± 11.5 mmHg for OD and OS, respectively. Conclusion: OPT was successfully adapted to the rabbit eye and elevated the IOP to pre-defined pressure values. This new and non-invasive procedure can be used as a transient rabbit glaucoma model system. It allows verification of new therapeutic interventions, like implanted drainage devices with minimal stress of the examined animals. It might also be of help to measure outflow facilities after application of new potentially IOP lowering medications.

Hintergrund: Die Abnahme der kornealen Hysterese(CH) bei Zunahme des Augeninnendruckes sowie eine größer werdende Differenz der ORA-Parameter IOPg (analog GAT) und IOPcc (korneal kompensierter IOD) im Vergleich zum Istdruck wurden in klinischen Studien beobachtet [2,4]. Daraus wurde die Vermutung abgeleitet, dass der Applanationsimpuls des ORA bei höherer Gewebespannung unzureichend wäre. Vor diesem Hintergrund evaluierten wir die diesbezüglichen spezifischen Parameter des ORA bei höherer Gewebespannung an einem experimentellen Augenmodell [3]. Methodik: Mit Hilfe der Steuerungseinheit eines biomechanischen Augenmodells wurde über zwei Schrittmotoren der "wahre" Intraokulardruck (Istdruck) in 10 Schweineaugen zwischen 10 und 40 mmHg geregelt. Bei stufenweiser Erhöhung und Absenkung des Istdruckes in 5 mmHg Schritten bestimmten wir den IOPg, IOPcc, den kornealen Resistenzfaktor (CRF) und die CH. In Abhängigkeit von der zentralen Hornhautdicke (CCT) wurde die Unterteilung in zwei Gruppen (Gr.1 < 1450μm, Gr.2 >1450μm) vorgenommen. Ergebnisse: Die Differenz IOPg und IOPcc zum Istdruck verhielt sich stets negativ. Der Unterschied IOPg/Istdruck betrug in der Gr.1 -8,0±2,8 mmHg und in Gr.2 -6,8±3,5 mmHg. Der IOPcc wurde weniger von der CCT beeinflusst (Gr.1: -2,4±4,1 mmHg; Gr.2: -3,1±4,0 mmHg). Die CH zeigt sich im gesamten Druckverlauf ohne statistisch signifikante Abweichung (Gr.1: 5,5±2,7 mmHg; Gr.2: 7,1±2,1 mmHg). Der CRF ließ eine direkt proportionale Abhängigkeit vom Istdruck erkennen und nahm mit steigendem Istdruck zu. Schlussfolgerung: Der IOPcc berücksichtigt Unterschiede in der CCT. Die CH zeigte trotz der Änderung des Istdruckes in aufsteigender als auch absteigender Reihenfolge keine relevante Veränderung. Die CH ist demnach von Istdruck und CCT weniger abhängig als vermutet. Nach den Messergebnissen zu urteilen, regeneriert der ORA ausreichend starke Applanationsimpulse. Aus der Sicht unserer Daten kann Alhamads T.A. et al. [1] Beurteilung bestätigt werden, dass die CH nicht allein von den Hornhauteigenschaften, sondern auch von weiteren biomechanischen Eigenschaften des Bulbus beeinflusst wird. Literatur: 1.Alhamad TA et al. (2011) Acta Ophtha 89:1755-3768 2.Neuburger M et al. (2010) Am J Ophtha 149:687-688 3.Saleh K et al. (2014) Biomed. Eng. Lett.:1-7 4.Sun L et al. (2009) Am J Ophtha 147:1061-1066

Introduction:The diagnostics of glaucoma is one of the most important problems in ophthalmology because of its great medical and social role and vision loss as an outcome of the disease. The prevalence of glaucoma is constantly increasing. To detect the eye pathology we test the visual field of each eye separately. But our perception that influences the quality of life is determined by two eyes. Binocular visual field testing and the study of quality of life is needed for the better understanding of the influence of open-angle glaucoma on patients. Objectives: To test monocular and binocular vusual fields, quality of life and compare them in patients with advanced open-angle glaucoma. Aims: To improve the management of glaucoma patients. Methods: We examined 38 patients (20 men, 18 women) with advanced (stage II) open-angle glaucoma in both eyes. We performed visual acuity evaluation, biomicroscopy, tonometry, gonioscopy, perimetry. Binocular visual fiend was studied with Esterman test that is based on the study of the function. The most important zones in Esterman test are in the middle and lower areas of the visual field, because the most important tasks (e.g. reading, walking) require the functioning of these areas. Quality of life was tested with Visual Function-14, which consists of 14 questions about the complexity of visual tasks, 7 of these questions estimate the quality of life that depends on the binocular visual field. Results:In the eyes with advanced open-angle glaucoma (stage II) there is 55%±4% of the normal monocular visual field, 4187%±0,53% of binocular visual field, 80%±4,75% of quality of life, 77%±4,37% (p< 0.05) of quality of life that depends on the binocular visual field preserved. Conclusions: The results of the study show the high level of adaptation to live with the disease and the necessity of the binocular visual field testing and assessment of the quality of life in all patients after 40 years who undergo ophthalmological examination.

Hintergrund: Der „okuläre Perfusionsdruck“ wurde in den letzten zwei Jahrzehnten ersatzweise als Differenz von Oberarmblutdruck und Augeninnendruck [IOP] berechnet. Mit dem Ocular Pressure Blood Flow Analyzer [OPFA] kann der pp_oc nichtinvasiv bestimmt werden. Die Ergebnisse der Abhängigkeit des pp_oc vom Systemblutdruck und vom Alter werden vorgestellt und ein Vergleich von gemessenen okulären Perfusionsdrucken und konventionell berechneten Daten wird vorgenommen. Material und Methoden: Das OPFA-Gerät [Hersteller: tpm Lüneburg] registriert nach artifizieller Erhöhung des IOP auf suprasystolische Werte bei steigendem pp_oc die okulären Pulsblutvolumina. Man erhält eine pp_oc-Pulsblutvolumenkurve, aus der die pp_oc abgelesen oder per Software erfasst werden. Von 118 Gesunden und 117 noch unbehandelten Patienten mit primärem Offenwinkelglaukom [POWG] wurden mit dem OPFA die okulären Perfusionsdrucke gemessen und den konventionell berechneten Daten gegenüber gestellt. Ein statistischer Vergleich wurde mit dem Wilcoxon Signed-Ranks Test vorgenommen. Die Abhängigkeit des mit dem OPFA gemessenen pp_oc vom Systemblutdruck und vom Alter wurde für die 118 Gesunden untersucht. Ergebnisse: Alle Vergleiche der mit dem OPFA gemessenen okulären Perfusionsdrucke mit den traditionell berechneten Daten zeigten sowohl bei den Gesunden als auch bei den Glaukompatienten statistisch hochsignifikante Differenzen. Der pp_oc ist vom Systemblutdruck und vom Alter abhängig. Schlussfolgerungen: Die okulären Perfusionsdrucke können nicht durch die traditionell berechneten sogenannten „okulären Perfusionsdrucke“ ersetzt werden. Die als Ersatz berechneten Daten sollten demzufolge zukünftig nicht mehr als okuläre Perfusionsdrucke bezeichnet werden. Sie sind ungeeignet, um Aussagen zur okulären Zirkulation zu machen. Die okuläre Zirkulation ist mit ihren autoregulatorischen Mechanismen darauf ausgerichtet, den pp_oc zur Sicherung der okulären Perfusion stets aufrecht zu erhalten.