Postersitzungen, Donnerstag, 29. 9. 2016

 
dt/engl
Posterkabinett 1 13:30 - 14:30 29.09.2016
Postersitzung PDo01
Glaukom: Grundlagen // Basics
Vorsitzende/r: Anselm Jünemann (Rostock), Thomas Klink (München)

Referent/in: Milena Pahlitzsch (London)
Introduction: Visualisation of Retinal Ganglion Cells (RGCs) using confocal scanning laser ophthalmoscopy is considered almost impossible due to their transparent nature. For this reason, the use of contrast agents to fluorescently label RGCs are increasingly used to distinguish RGCs and pathologies. The aim of this study is to evaluate the in vivo apoptosis of RGCs using intravenously administered fluorescently labelled Annexin A5 (Anx776). Methods: An in vivo rat model of N-methyl-D-aspartate (NMDA)-induced neurotoxicity was used (n=20, unilateral dosing) and 10 healthy rats (n=20 eyes) were examined as controls without NMDA insult. Adult male rats received an intravitreal injection of 40mM NMDA in the left eye. 24 hours later intravenous administration of Anx776 was given. Retinal imaging was performed 2 hours post Anx776 administration using a confocal scanning laser ophthalmoscope (cSLO, Spectralis Heidelberg Engineering, Germany). Individual fluorescently labelled RGCs were then manually counted from retinal images by 3 masked operators with apoptotic retinal cell counts and inter-observer variability determined. In addition, an in vivo safety profile was assessed prior to the NMDA experiment. Results: NMDA induced apoptosis in the retina. In all Anx776 dosed groups a significant (p< 0.05, two-way ANOVA) increase in the number of apoptotic RGCs was detected in eyes which received NMDA versus control eyes. A dose dependent variation in the number of apoptotic retinal cells was observed. RGC counts were found to correlate well between three masked operators with a high inter-observer agreement reported (Pearson r >0.9, p< 0.05). Anx-776 administered by intravenous injection to the rat up to the maximum study dose of 0.29 mg/kg/day was found to be well tolerated, with no treatment-related changes in blood chemistry, haematology and coagulation, ophthalmoscopy, food intake or body or organ weights. Conclusion: Retinal cell apoptosis can be detected at a cellular resolution after intravenous administration of Anx776 contrast agent and no safety concerns were raised by in vivo toxicity assessment. A significant increase in apoptotic retinal ganglion cells was observed in a rat model of NMDA neurotoxicity, suggesting this contrast agent may have utility for the diagnosis of retinal disorders after intravenous administration.
Referent/in: Nauke Zeleny (Aarau)
Background: To investigate on the aqueous humor proteome in patients with glaucoma and a control group. Material and Methods: Aqueous humor was obtained from five human donors diagnosed with primary open angle glaucoma (POAG) and five age- and sex-matched controls undergoing cataract surgery. Quantitative proteome analysis of the aqueous humor by hyper reaction monitoring mass spectrometry (HRM-MS) based on SWATH technology was performed. Results: Expression levels of 87 proteins were found to be different between glaucomatous and control aqueous humor. Of the 87 proteins, 34 were significantly upregulated, whereas 53 proteins were downregulated in the aqueous humor from glaucoma patients compared to controls. Differentially expressed proteins were found to be involved in cholesterol-related, inflammatory, metabolic, antioxidant as well as proteolysis-related processes. Conclusions: Glaucoma leads to profound changes in the aqueous humor proteome consistent with an altered metabolic state, an inflammatory response and impaired antioxidant defense.
Referent/in: Stephanie Joachim (Bochum)
Purpose: As previously shown, immunization with ocular antigens, like heat shock protein 27 (HSP27), leads to RGC loss in an autoimmune glaucoma model. Aim of this study was to investigate, how immunization with S100 alone and in combination with HSP27 affects neuronal and glial cells in the retina at later stages. Methods: Rats were immunized with S100 or S100 plus HSP27 (COMB) and compared to controls (n=5/group). 4 weeks after immunization, retinas were processed for immunehistology and Western blot analysis. Retinal ganglion cells (Brn-3a), amacrine cells (ChAT), and photoreceptors (rhodopsin, opsin) were quantified. Additionally, macroglia (GFAP, Vimentin) were analyzed. Groups were compared via ANOVA with Dunnett’s post-hoc test. Results: No IOP alterations were noted in both immunized groups throughout the study (both: p>0.05). About a 30% RGC loss was observed in both immunized groups at 4 weeks (S100 p=0.005; COMB p=0.004). Cholinergic amacrine cells were also affected (S100 p=0.02; COMB p=0.045), while cone (S100 p=0.5; COMB p=0.2) and rod photoreceptors (S100 p=0.9; COMB p=0.1) remained intact in both immunized groups. A slight increase in astrocyte reactivity was noted in both immunized groups (S100 p=0.05; COMB p=0.04), while Müller glia remained unaltered (both: p=0.9). Discussion: Findings from this study indicate that immunization with ocular antigens rather damages RGCs and amacrine cells than photoreceptors. Hence, this IOP-independent glaucoma model can serve as a tool to study specific neuroprotective agents.
Referent/in: Hanhan Liu (Mainz)
Purpose: Hydrogen sulfide (H₂S) is a gas endogenously produced within endothelial cells. It represents a novel third signaling molecule, neurotransmitter and cytoprotectant. Recently, cell protective properties within the central nervous system and cardiovascular system have been proposed. However, its role within the pathogenesis of glaucoma remains completely unknown. Purpose of our study was to 1) analyse the expression changes of H₂S in an experimental animal glaucoma model and 2) to analyze its potential neuroprotective effect on retinal ganglion cells (RGC) against cell death due to elevated pressure in vitro and in vivo. Methods: Experimental glaucoma was induced in adult Sprague Dawley rats by cauterization of three episcleral veins of the left eye (n=23). Animals were sacrificed after 7 weeks of IOP elevation.Changes in H₂S and 3-mercaptopyruvate, one of the three key enzymes producing H₂S endogenously, expression within the retina was analyzed via label-free mass spectrometry. In vitro, organotypic retinal explants were cultured for two days without elevated pressure or under controlled elevated hydrostatic pressure within a high-pressure chamber (60 mmHg) with and without addition of different concentrations of GYY4137 (1 nM-10 μM), a slow-releasing H₂S donor to medium. Subsequently surviving RGCs were immunohistochemically stained against BRN3A in retinal flat mounts, counted and compared within the groups. Furthermore H₂S in the best concentration was injected intravitreally in animals with elevated IOP and RGC survival was measured by BRN3A staining. RNFL thickness and vessel diameters were measured in vivo by Spectralis-OCT (Heidelberg Engineering). Multiple comparisons were made by ANOVA. p< 0.05 was set as statistically significant. Results: In cauterized eyes (p< 0.0001) IOP was significantly increased and we observed a significant ganglion cell loss over time (p< 0.0001). Using proteomics H₂S showed a significant downregulation after 7 weeks of elevated intraocular pressure compared to controls (p< 0.05) and 3-mercaptopyruvate sulfurtransferase a significant upregulation in retina of glaucomatous animals. Application of H₂S to organotypic retinal cultures showed a dose dependant neuroprotective effect. Within the concentration range of 1 nM to 100 nM,GYY4137 could significantly improve RGC survival under elevated hydrostatic pressure in vitro (p< 0.005), while higher doses remained toxic. In vivo intravitreal addition of H₂S preserved RGCs from IOP induced cell death. Vessel diameters around the optic nerve head measured by OCT appeared enlarged. Conclusion: H₂S seems to play a key role in experimental glaucoma. Addition of H₂S protects RGC from cell death due to elevated hydrostatic pressure in vitro and in vivo possibly by affecting the endothelial cells. The upregulation of 3-mercaptopyruvate sulfurtransferase in retina of glacomatous animals is possibly due to cellular self-protection mechanism against glaucomatous damage. Keyword:hydrogen sulfide,retinal ganglion cell,glaucoma,neuroprotection
Referent/in: Charlotte Viola Fischer (Göttingen)
Fragestellung: Die subkonjunktivale Applikation des Vascular Endothelial Growth Factor (VEGF)- Inhibitors Bevacizumab (BVC) stellt einen neuen Ansatz zur Vernarbungshemmung nach filtrierender Glaukom-Operation dar. Es wurde untersucht, ob es sich dabei um einen VEGF-vermittelten oder Antigen-unabhängigen Effekt handelt. Methoden: Primäre Zellkulturen von humanen Tenon-Fibroblasten (hTF) wurden mit BVC (2,5 bis 10 mg/ml), Ranibizumab (RNB) (2,5mg/ml), Aflibercept (AFB) (5 und 10mg/ml) und dem CD20-Antikörper Rituximab (RTX) (2,5 und 5mg/ml) behandelt. Die Gesamtzellzahl, und die Anzahl toter Zellen wurde nach 24h bestimmt (LIVE/DEAD ® cell imaging kit). Intrazelluläre IgG-Aufnahme wurde durch Immunhistochemie und WesternBlot, VEGF-Konzentrationen mittels ELISA gemessen. Antikörper und Lösungsmittel von BVC wurden mittels Filtration getrennt. Ergebnisse: BVC erniedrigte signifikant die Gesamtzellzahl und erhöhte die Anzahl toter Zellen nach 24h in Konzentrationen höher als 5mg/ml (Gesamtzahl: 1,2±0,8 (MW+SEM) vs. 32,4±2,3 /mm2; p< 0,0001; tote Zellen: 14.8±1.1 vs. 3.7±1.2 /mm2; p< 0,001). Ebenso wirkte die Antigen-unabhängige Behandlung mit RTX in Konzentrationen höher als 2,5mg/ml (Gesamtzahl: 21.9±4.5 vs. 50.7±10.1 /mm2; p=0.026; tote Zellen: 11.4±2.1 vs. 3.1±1.2 /mm2; p=0,0061). AFB (10mg/ml) und RNB (2,5mg/ml) hatten keinen Einfluss auf die Zellzahl und Zelltod (Gesamtzahl: 32,4±4,1 (AFB) bzw. 35,4±2,8 (RNB) vs. 32,4±2,3 /mm2; tote Zellen: 4,7±2,0 (AFB) bzw. 4,6±0,8 (RNB) vs. 3,7±1.2 /mm2). Die Behandlung mit antikörperfreier BVC-Lösung erhöhte die Zahl toter Zellen, erniedrigte aber nicht die Gesamtzellzahl (16,3±3,4 vs. 4,6±0,9 tote Zellen/mm2; p=0,0085). Die Konzentration von freiem VEGF 165 im Kulturmedium wurde von BVC und AFB auf Werte unterhalb der Nachweisgrenze reduziert. Intrazelluläres IgG konnte konzentrationsabhängig nach der Behandlung mit BVC, AFB und RTX nachgewiesen werden. Schlussfolgerung: Obwohl BVC und AFB gleich effektiv VEGF inhibieren, bewirkt nur BVC einen Zellverlust. Hingegen zeigt der CD20-Antikörper RTX eine vergleichbare Wirkung wie BVC. Dies legt einen VEGF-unabhängigen Effekt von BVC auf hTF nahe.
Referent/in: Susanne Wiemann (Bochum)
Purpose: Glaucoma disease is characterized by the degeneration of optic nerve fibers and death of retinal ganglion cells (RGCs). Intraocular pressure (IOP) elevation is a main risk factor for glaucoma. The molecular mechanisms of this disease are not fully understood yet. Mice heterozygous (HET) for the protein tyrosine phosphatase Meg2 (PTP-Meg2) develop a significant IOP elevation and RGC loss. In the present study, we investigate glial response and remodeling of the extracellular matrix glycoprotein Tenascin-C (TNC) in the glaucomatous PTP-Meg2 HET mouse model. Methods: IOP was measured by tonometry in PTP-Meg2 (HET) and wildtype (WT) mice. The number of Brn3a+ RGCs and Iba1+ microglia was quantified in retinal whole mount explants. Iba1+ cells were also analyzed in optic nerve slices. Additionally, GFAP+ macroglia and the ECM component TNC were examined in retina and optic nerve cross sections. Groups were analyzed via Students t-test. Results: A significant IOP increase (p< 0.001) was observed after 28 weeks in PTP-Meg2 HET mice. At this point in time a significant loss of RGCs was detected (p=0.007). Also, HET mice showed a significantly increased number of Iba1+ microglia in the retina (p=0.011) and optic nerve (p=0.04) in comparison to WT. In the central (p=0.03) as well as in the peripheral (p=0.005) part of the retina microglia number was significantly elevated in HET mice. GFAP immunoreactivity was significantly increased in the whole mount retina (p< 0.001) and optic nerve (p=0.04) of PTP-Meg2 HET mice. Furthermore, TNC immunoreactivity was significantly upregulated in the retina (p=0.0003) and optic nerve (p=0.03) of HET in comparison to WT mice. Conclusion: In the present study we verified IOP elevation and RGC loss in PTP-Meg2 HET mice. Furthermore, we showed an increased number of microglia as well as an increased GFAP and TNC immunoreactivity in the retina and optic nerve. In conclusion, progressive IOP elevation and RGC loss are associated with an increased glial reactivity and remodeling of the ECM component TNC in the retina and optic nerve of PTP-Meg2 HET mice.
Referent/in: Carolina Mann (Mainz)
Introduction: Glaucoma is characterized by progressive retinal ganglion cell and axonal loss. 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 intraocular pressure (IOP). Aim of this study was to detect IOP induced vascular changes in the vessels of the optic nerve head and the main vessels of the retina in vivo and in primary isolated endothelial cells in vitro. Methods: Experimental glaucoma was induced in adult Sprague Dawley rats by cauterization of three episcleral veins of the left eye (n=6). In vivo, retinal vessel calibre was manually measured using a peripapillary scan with SD-OCT (HeidelbergEngineering) after six weeks of IOP elevation. Following animals were sacrificed and the optic nerve was fixed with 30% glutaraldehyd and cross sections stained with paraphenylendiamine to mark vessels. Contralateral eyes served as controls. Pictures were taken and number of vessels, vessel calibre and area was calculated and compared. In vitro, dissociated brain endothelial cells were exposed to normal pressure (control) or to elevated pressure (60 mmHg) within a high pressure incubation chamber for two days. A life-death assay was performed to allow cell viability measurement. In vitro, organotypic retinal explants were cultured under normal conditions over two days or within the high pressure chamber (60 mmHg), flatmounted, and stained immunohistochemically against Endothelin-1 to mark endothelial cells. Results: IOP could be significantly elevated (p< 0.001). In optic nerve cross sections the number of capillaries did not significantly change between animals with elevated IOP and controls. However, vessel calibre and area was significantly reduced by 1.723 µm (12.2%, p< 0.0001) and 2.357 µm² (18.7%, p< 0.001) respectively in glaucomatous optic nerves. The calibre of the retinal vessels was significantly lowered by 9.22% as well (p=0.021). In vitro, capillary endothelial cells responded to elevated pressure by significantly (p< 0.001) decreased viability and cell death. In retinal explants endothelial cells could not be stained immunohistochemically after two days under elevated pressure at all. Conclusion: Retinal capillaries respond sensitively to abnormal pressure-elevation both in vivo and in vitro, showing a high and early vulnerability. The capillary responses may influence secondary neuronal responses which culminate in death of ganglion cells and blindness as occurs in clinical glaucoma.
Referent/in: Katharina Bell (Darmstadt)
Purpose: Glaucoma is a multifactorial, neurodegenerative disease and previous studies were able to detect several up- as well as down-regulated autoantibodies in the serum of glaucoma patients. We already were able to demonstrate protective effects of the antibody (ab) against GFAP, which is down-regulated in glaucoma patients, on immortalized neuroretinal cells. With this study we aimed to analyse the protective antibody effect in more detail using a porcine retinal organ culture. Material and methods: The retina along with the RPE was isolated from a porcine eye derived from 3-6 month old pigs. The retinal explants were placed in transwells and incubated either with medium containing 1 µg/ml GFAP antibodies for 24 h or without antibodies as control. Mass spectrometric analysis of the retinae were conducted. Following incubation Brn3a, DAPI, TUNEL and glutamine synthetase staining were performed. Retinal ganglion cell quantification was performed as well as an analysis of the glutamine synthetase intensity. Glutamine synthetase analysis was performed by measuring the overall staining in the retinae as well as the intensity of staining in the retinal ganglion cell layer. Results: Rgc/mm increased significantly in GFAP ab incubated retinal explants (17.9 rgc/mm) (p=0.04) in comparison to the untreated controls (13.8 rgc/mm). Mass spectrometric analysis revealed elevated glutamine synthetase levels (2.5 fold upregulated in the GFAP ab incubated retinae). The overall intensity of glutamine synthetase in the retinae was slightly elevated in the GFAP group. When then analyzing the glutamine synthetase staining in the retinal ganglion cell layer, significant differences between the ab incubated retinae and the control retinae (Intensity control retinae: 58.05; GFAP ab treated retinae: 85.96) (p< 0.05) were detected. Conclusions: Low concentrations of GFAP antibodies have a protective effect on retinal ganglion cells in a retinal explant culture. We believe that Müller cells participate in the demonstrated protective ab effects leading to a shift of glutamine synthetase towards the inner border of the retina. This could be either triggered via direct antibody/müller cell contact or indirectly via rgc/müller cell contact.
Referent/in: Dirk Bauer (Münster)
Fragestellung: Das Sekundärglaukom bei Uveitis ist eine häufig auftretende Komplikation, die unbehandelt zu einem signifikanten Funktionsverlust oder zur Erblindung führen kann. In dieser Studie untersuchten wir den Einfluss von erhöhtem hydrostatischem Druck auf Makrophagen. Methodik: Knochenmarkmakrophagen von B10.RIII Mäusen wurden mittels Kokultur mit macrophage colony-stimulating factor (M-CSF)-haltigem Überstand gewonnen und mit verschiedenen Drücken (Raum-, 20 mmHg und 60 mmHg -Druck) und unterschiedlichen Lipopolysaccharid (LPS) Konzentrationen für 48h inkubiert. Die Zytokinkonzentrationen in den Zellkulturüberständen wurden mittels Bioassay (IL-1, IL-6, TNF-a und NO) oder ELISA (IL-6, IL-12 und TNF-a) untersucht. Die Viabilität von Makrophagen und auch Lymphozyten (Splenozyten) wurde mittels MTT Test bestimmt. Ergebnisse: Nach Kultur der Makrophagen unter erhöhtem hydrostatischem Druck zeigte sich eine erhöhte Menge von TNF-a und IL-12 und eine erhöhte biologische Aktivität von IL-6 und TNF-a im Kulturüberstand. Die Produktion von NO war nach Kultivierung unter höherem hydrostatischem Druck deutlich vermindert. Makrophagen zeigten im Gegensatz zu Lymphozyten unter erhöhtem hydrostatischen Druck kaum eine Reduktion der Viabilität. Schlussfolgerungen: Makrophagen haben im Vergleich zu Lymphozyten unter höherem hydrostatischem Druck deutliche Überlebensvorteile. Makrophagen zeigen nach Druckerhöhung eine Zellantwort, die auch bei Patienten mit Glaukom gefunden wurde.
Referent/in: Michael Böhm (Essen)
Purpose: To determine the role of high-mobility group box 1 protein (HMGB-1) induced neurodegeneration and inflammation in photoreceptors exposed to elevated pressure in an experimental glaucoma model in-vitro. Methods: Mouse retinal photoreceptor-derived cells (661W) and retinal explants naturally containing photoreceptors were incubated under elevated pressure to examine whether recombinant HMGB- 1 (rHMGB-1) is associated with inflammatory or degenerative events. Immunohistochemistry, Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to examine the expression levels of HMGB-1, receptor for advanced glycation end products (RAGE), toll-like receptors 2 and 4 (TLR-2, TLR-4), apoptosis-related factors (e.g., B-cell lymphoma 2 (Bcl-2), Bcl-2- associated death promoter (Bad). Release of cytokines, like tumor necrosis factor-α (TNF-α), interleukin (IL)-4, IL-6, and vascular endothelial growth factor (VEGF) were examined by a cytokine array. Occurrence of apoptosis was revealed by TUNEL assay. Results: The data revealed an increased expression of HMGB-1, RAGE, TLR-2 and -4, cytokines (e.g.,TNF-α) and apoptotic factors in 661W cells exposed to elevated pressure. Addition of HMGB-1 to the 661W cells resulted in apoptosis and induced an increased release of cytokines (e.g. TNF-α, IL-4, IL-6, and VEGF). An up-regulation of HMGB-1, TLR-2 and RAGE as well as anti-apoptotic Bcl-2 expressions were found in the retinal explants exposed to elevated pressure and rHMGB-1 indicating a switch of the retinal homeostasis to a pro-inflammatory and pro-survival state. Conclusions: The results suggest that HMGB-1 promotes an inflammatory response and mediates apoptosis in the pathology of photoreceptors and retinal homeostasis. HMGB-1 may play a key role in ongoing damage of retinal cells under conditions of elevated pressure around the cells or the entire retina as this naturally occurs within the eye under glaucomatous conditions.
Referent/in: Viktoria Mans (Göttingen)
Fragestellung: Zellen sind in der Lage die Elastizität ihres Untergrundes wahrzunehmen. Diese wiederum beeinflusst maßgeblich ihre Antwort auf Umgebungsreize. Die physiologische Umgebung von humanen Tenon-Fibroblasten (hTF) ist deutlich weicher als Standard-Zellkulturplatten. Wir vermuten, dass allein die Kultivierung auf physiologisch weichen Gelen eine Auswirkung auf die Reaktion auf verschiedene Pharmaka hat. Methode: Die Elastizität humanen Tenongewebes von erwachsenen Spendern wurde mittels Atomic Force Microscopy (AFM) bestimmt (n=3). Aus den Tenonproben wurden Primärkulturen von hTF angelegt, welche auf unterschiedlich harten Polyacrylamid-Geluntergründen kultiviert wurden (0,75 bis 30 kPa). Nach 24 Stunden Kultivierungsdauer unter 0,2% Serum wurde das Zytoskelett mit Phalloidin und der Kern mit DAPI gefärbt und sowohl die Morphologie der Zellen mittels ImageJ-Software quantifiziert (n=18), als auch die Proliferation auf Basis der Zellanzahl ermittelt (n=3). Die Wirkung von Diclofenac (0,01%; 12 h), Dexamethason (0,01%; 12 h) und Mitomycin C (0,2 mg/ml; 5 min) wurde auf Gelen der Elastizität 2kPa sowie auf Glas getestet (n=3 pro Gruppe). Die Anzahl lebender und toter Zellen wurde mittels Vitalfärbung (Life/Dead Kit) bestimmt. Die statistische Auswertung erfolgte mittels ANOVA. Resultate: Die durch AFM ermittelte Elastizität humanen Tenongewebes von erwachsenen Spendern rangierte zwischen 2 kPa und 7 kPA (Mittel 4 kPA). Die Zellform wurde bei weicheren Geluntergründen runder (Circularity-Index 0,75 vs. 30 kPA: 13,6±8,8 vs. 38,2±16,1; p< 0,0001) und die Zellzahl zeigte eine sinkende Tendenz (2 kPa vs Glas: 35±18 vs 67±16; p=0,08). In der Medikamententestung auf 2 kPa Gel führte nur Mitomycin C zu einer signifikanten Abnahme der Zellzahl (p< 0.05). Auf Glas reduzierten Voltaren und Mitomycin C die Zellzahl (p< 0,05 und p< 0,01). Im Vergleich zwischen 2 kPa Gelen und Glasuntergrund war die Wirksamkeit aller drei Medikamente nicht unterschiedlich (ANOVA). Auf 2 kPa Gelen zeigte sich nach Behandlung mit Diclofenac eine gegenüber Glas signifikant erhöhte Anzahl toter Zellen (p=0.025) Schlussfolgerung: Die Kultivierung von hTF auf physiologisch weichem Geluntergrund führt zu einer signifikanten Änderung der Zellmorphologie und tendenziell geringerer Proliferation. Ob ein Einfluss des Untergrundes auf die Wirkung von Medikamenten besteht, konnte nicht eindeutig belegt werden.
Referent/in: Fabian Anders (Mainz)
Purpose: Glaucoma is characterized by progressive irreversible retinal ganglion cell loss (RGC). Increased intraocular pressure (IOP) is considered as a main risk factor. However, the molecular changes remain obscure and the disease progresses despite significant IOP reduction. Aim of this study was to explore the retinal proteome in an in vivo glaucoma model to analyze molecular mechanisms and explore neuroprotective treatments by reversing these changes. Methods: IOP was increased in the left eye of each Sprague-Dawley rat by cauterization of three episcleral veins (n=23). Animals were sacrificed after 3 or 7 weeks of IOP elevation. RGC count was conducted by immunohistological staining against ganglion cell specific BRN3A in retinal flat mounts, axon count in optic nerve cross sections and decrease of the retinal nerve fiber layer was investigated via optical coherence tomography (SD-OCT, Heidelberg Engineering) in vivo. Proteomic changes, especially in the crystallin family, within the retina and the vitreous body were analyzed via label-free mass spectrometry. As βB2-crystallin turned out to be down-regulated at three weeks after IOP rise, 10 µg βB2-crystallin was injected intravitreally into the vitreous body of OS eyes with beginning of IOP rise (n=8). Results: IOP was increased to 17.83±0.83 mmHg in the OS eye, while the fellow eye remained at 11.27±0.28 mmHg (p< 0.0001). There was a significant decrease in RGC and axon number as well as RNFL thickness due to elevated IOP (p< 0.0001). Using proteomics, the crystallin family showed a specific IOP related change. βB2-Crystallin induced a significant down-regulation in the treated eye with a 5.25-fold-change (p< 0.05) after three weeks of elevated IOP, followed by an up-regulation in the 7 weeks control animals (FC=2.6). Injection of βB2-Crystallin prior to IOP elevation reduced the RGC loss by 12% and the RNFL loss by 11% compared to the control groups (p< 0.0001). Cross sections of the optic nerve showed an axonal damage exposed to elevated IOP, which could be also reduced by prior crystallin injection. Conclusion: βB2-Crystallin was significantly down-regulated after three and up-regulated after 7 weeks of IOP elevation . This long-considered lens specific protein seems to have a specific impact on molecular changes in glaucoma. We suspect the down-regulation to be a reaction to the acute IOP elevation, followed by a delayed up-regulation as a cellular protection mechanism. Intravitreal injection of βB2-Crystallin prior to IOP elevation enhanced RGC cell survival tremendously. Thus, βB2-Crystallin might be a promising candidate to protect from glaucomatous damage also in human glaucoma.