Rat retinae data for use as spectral library, for pathway remodeling as well as protein mapping.

This article describes a mass spectrometric data set from rat retinae spiked with indexed Retention Time (iRT) peptides. The provided data set can be used as a spectral library to investigate for instance eye disorders as well as ocular function by data-independent acquisition (DIA) based mass spectrometry. Consequently, there is no urgent need to create an own spectral library, which requires money, time, effort as well as tissue. Besides the use as a spectral library, this data set can improve our knowledge about proteins present in the rat retina and thus the protein pathways within this tissue. The data set may also help to determine optimal parameters for peptide identification by mass spectrometry. To generate the presented data set, six rat retinae were homogenized with glass beads and pooled. The pooled sample was fractionated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) followed by tryptic in-gel digestion. The fractionation of the pooled sample was repeated for further 4 times, to end up with in total 5 technical replicates. Peptide extracts were spiked with iRT peptides and analyzed by data-dependent (DDA) nanoHPLC-ESI-MS/MS resulting in 60 files. All resulting data files are hosted in the public repository ProteomeXchange under the identifier PXD021937.

Reduced Retinal Degeneration in an Oxidative Stress Organ Culture Model through an iNOS-Inhibitor.

In retinal organ cultures, H2O2 can be used to simulate oxidative stress, which plays a role in the development of several retinal diseases including glaucoma. We investigated whether processes underlying oxidative stress can be prevented in retinal organ cultures by an inducible nitric oxide synthase (iNOS)-inhibitor. To this end, porcine retinal explants were cultivated for four and eight days. Oxidative stress was induced via 300 µM H2O2 on day one for three hours. Treatment with the iNOS-inhibitor 1400 W was applied simultaneously, remaining for 72 h. Retinal ganglion cells (RGC), bipolar and amacrine cells, apoptosis, autophagy, and hypoxia were evaluated immunohistologically and by RT-qPCR. Additionally, RGC morphology was analyzed via transmission electron microscopy. H2O2-induced RGCs loss after four days was prevented by the iNOS-inhibitor. Additionally, electron microscopy revealed a preservation from oxidative stress in iNOS-inhibitor treated retinas at four and eight days. A late rescue of bipolar cells was seen in iNOS-inhibitor treated retinas after eight days. Hypoxic stress and apoptosis almost reached the control situation after iNOS-inhibitor treatment, especially after four days. In sum, the iNOS-inhibitor was able to prevent strong H2O-induced degeneration in porcine retinas. Hence, this inhibitor seems to be a promising treatment option for retinal diseases.

Proteomic Analysis of Retinal Tissue in an S100B Autoimmune Glaucoma Model.

Glaucoma is a neurodegenerative disease that leads to damage of retinal ganglion cells and the optic nerve. Patients display altered antibody profiles and increased antibody titer, e.g., against S100B. To identify the meaning of these antibodies, animals were immunized with S100B. Retinal ganglion cell loss, optic nerve degeneration, and increased glial cell activity were noted. Here, we aimed to gain more insights into the pathophysiology from a proteomic point of view. Hence, rats were immunized with S100B, while controls received sodium chloride. After 7 and 14 days, retinae were analyzed through mass spectrometry and immunohistology. Using data-independent acquisition-based mass spectrometry, we identified more than 1700 proteins on a high confidence level for both study groups, respectively. Of these 1700, 43 proteins were significantly altered in retinae after 7 days and 67 proteins revealed significant alterations at 14 days. For example, α2-macroglobulin was found significantly increased not only by mass spectrometry analysis, but also with immunohistological staining in S100B retinae at 7 and 14 days. All in all, the identified proteins are often associated with the immune system, such as heat shock protein 60. Once more, these data underline the important role of immunological factors in glaucoma pathogenesis.

Minocycline reduces inflammatory response and cell death in a S100B retina degeneration model.

BACKGROUND: Previous studies noted that intravitreal injection of S100B triggered a glaucoma-like degeneration of retina and optic nerve as well as microglia activation after 14 days. The precise role of microglia in our intravitreal S100B model is still unclear. Hence, microglia were inhibited through minocycline. The aim is to investigate whether microglia have a significant influence on the degeneration process or whether they are only a side effect in the model studied here. METHODS: Minocycline was applied daily in rats by intraperitoneal injection using two different concentrations (13.5 mg/kg body weight, 25 mg/kg body weight). One day after treatment start, S100B or PBS was intravitreally injected in one eye per rat. The naïve groups received no injections. This resulted in a total of five groups (naïve n = 14, PBS n = 14, S100B n = 13, 13.5 mg/kg mino n = 15, 25 mg/kg mino n = 15). At day 14, electroretinogram measurements were performed, followed by immunofluorescence and label-free quantitative proteomics analysis. The focus of these investigations was on the survival of RGCs as well as their axons, the response of the microglia, and the identification of further pathological modes of action of S100B. RESULTS: The best signal transmission was detected via ERG in the 13.5 mg/kg mino group. The inhibition of the microglia protected optic nerve neurofilaments and decreased the negative impact of S100B on RGCs. However, the minocycline treatment could not trigger complete protection of RGCs. Furthermore, in retina and optic nerve, the minocycline treatment reduced the number and activity of S100B-triggered microglia in a concentration-dependent manner. Proteomics analysis showed that S100B application led to numerous metabolic functions and cellular stress, mainly an increased inflammatory response, glycolysis, and mitochondrial dysfunction, which caused oxidative stress in the retina. Importantly, the protective capability of lower dose of minocycline was unraveled by suppressing the apoptotic, inflammatory, and the altered metabolic processes caused by S100B insult in the retina. CONCLUSION: Intravitreally injected S100B not only led to a pro-inflammatory microglial reaction, but also a mitochondrial and metabolic dysfunction. Also, these results suggest that an excessive microglial response may be a significant degenerative factor, but not the only trigger for increased cell death.

Synapse and Receptor Alterations in Two Different S100B-Induced Glaucoma-Like Models.

Glaucoma is identified by an irreversible retinal ganglion cell (RGC) loss and optic nerve damage. Over the past few years, the immune system gained importance in its genesis. In a glaucoma-like animal model with intraocular S100B injection, RGC death occurs at 14 days. In an experimental autoimmune glaucoma model with systemic S100B immunization, a loss of RGCs is accompanied by a decreased synaptic signal at 28 days. Here, we aimed to study synaptic alterations in these two models. In one group, rats received a systemic S100B immunization (n = 7/group), while in the other group, S100B was injected intraocularly (n = 6-7/group). Both groups were compared to appropriate controls and investigated after 14 days. While inhibitory post-synapses remained unchanged in both models, excitatory post-synapses degenerated in animals with intraocular S100B injection (p = 0.03). Excitatory pre-synapses tendentially increased in animals with systemic S100B immunization (p = 0.08) and significantly decreased in intraocular ones (p = 0.04). Significantly more n-methyl-d-aspartate (NMDA) receptors (both p ≤ 0.04) as well as gamma-aminobutyric acid (GABA) receptors (both p < 0.03) were observed in S100B animals in both models. We assume that an upregulation of these receptors causes the interacting synapse types to degenerate. Heightened levels of excitatory pre-synapses could be explained by remodeling followed by degeneration.

Protective effect of the extremolytes ectoine and hydroxyectoine in a porcine organ culture.

PURPOSE: Hypoxic damage to the retina is a relevant component of neurodegenerative pathologies such as glaucoma or retinal ischemia. In porcine retina organ cultures, hypoxic damage can be induced by applying cobalt chloride (CoCl2). The aim of our study was to investigate possible neuroprotective effects of the extremolytes ectoine and hydroxyectoine in this hypoxia-damaged retina model. METHODS: To simulate hypoxia, porcine retina organ cultures were damaged with 300 µM CoCl2 for 48 h starting on day 1 (n = 8-9/group). In order to investigate the possible neuroprotective effects of ectoine and hydroxyectoine, 0.5 mM of each extremolyte was added to the culture at the same time as the stressor and for the same duration. On day 8, the retina organ cultures were taken for (immuno)-histochemical examinations. Retinal ganglion cells (RGCs), macroglia, and apoptotic and hypoxic cells were detected with appropriate markers followed by cell counts and group comparisons. RESULTS: Treatment with ectoine resulted in RGC protection (p < 0.05) and reduced rate of apoptosis (p < 0.001) in hypoxia-treated retina organ cultures. However, the macroglia area and the amount of hypoxic, HIF-1α+ cells were unaffected by the ectoine treatment (p = 0.99). Treatment with hydroxyectoine also protected RGCs (p < 0.01) by inhibiting apoptosis (p < 0.001). In addition, the number of hypoxic, HIF-1α+ cells could be significantly reduced by treatment with hydroxyectoine (p < 0.05). The macroglia area on the other hand was unchanged after CoCl2 and treatment with hydroxyectoine. CONCLUSION: Both extremolytes had a protective effect on CoCl2-induced hypoxia in the porcine retina organ culture. Regarding the reduction of hypoxic stress, hydroxyectoine appears to be more effective. Thus, both extremolytes represent an interesting potential new therapeutic approach for patients with ocular diseases in which hypoxic processes play a significant role.

iNOS-inhibitor driven neuroprotection in a porcine retina organ culture model.

Nitrite oxide plays an important role in the pathogenesis of various retinal diseases, especially when hypoxic processes are involved. This degeneration can be simulated by incubating porcine retinal explants with CoCl2 . Here, the therapeutic potential of iNOS-inhibitor 1400W was evaluated. Degeneration through CoCl2 and treatment with the 1400W were applied simultaneously to porcine retinae explants. Three groups were compared: control, CoCl2 , and CoCl2  + iNOS-inhibitor (1400W). At days 4 and 8, retinal ganglion cells (RGCs), bipolar, and amacrine cells were analysed. Furthermore, the influence on the glia cells and different stress markers were evaluated. Treatment with CoCl2 resulted in a significant loss of RGCs already after 4 days, which was counteracted by the iNOS-inhibitor. Expression of HIF-1α and its downstream targets confirmed the effective treatment with 1400W. After 8 days, the CoCl2 group displayed a significant loss in amacrine cells and also a drastic reduction in bipolar cells was observed, which was prevented by 1400W. The decrease in microglia could not be prevented by the inhibitor. CoCl2 induces strong degeneration in porcine retinae by mimicking hypoxia, damaging certain retinal cell types. Treatment with the iNOS-inhibitor counteracted these effects to some extent, by preventing loss of retinal ganglion and bipolar cells. Hence, this inhibitor seems to be a very promising treatment for retinal diseases.

Oxidative stress-induced retinal damage is prevented by mild hypothermia in an ex vivo model of cultivated porcine retinas.

BACKGROUND: Hydrogen peroxide (H2 O2 ) can be used in vitro to simulate oxidative stress. In retinal organ cultures, H2 O2 induces strong neurodegeneration of the retina. It is known that oxidative stress plays a role in the development of several retinal diseases including glaucoma and ischemia. Thus, we investigated whether processes underlying oxidative stress can be prevented by hypothermia using an ex vivo organ culture model of porcine retinas. METHODS: Porcine retinal explants were cultivated for 5 and 8 days. Oxidative stress was induced via 300 µM H2 O2 on day 1 for 3 hours. Hypothermia treatment at 30°C was applied simultaneously with H2 O2 , for 3 hours. Retinal ganglion cells (RGCs), apoptosis, bipolar and cholinergic amacrine cells, microglia and macroglia were evaluated immunohistologically. Apoptosis rate was additionally analysed via western blot. RESULTS: Reduced apoptosis rates through hypothermia led to a preservation of RGCs (P < .001). Amacrine cells were rescued after hypothermia treatment (P = .17), whereas bipolar cells were only protected partly. Additionally, at 8 days, microglial response due to oxidative stress was completely counteracted via hypothermia (P < .001). CONCLUSIONS: H2 O2 induced strong degenerative processes in porcine retinas. The role of oxidative stress in the progression of retinal diseases makes this ex vivo organ culture model suitable to investigate new therapeutic approaches. In the present study, the damaging effect of H2 O2 to several retinal cell types was counteracted or strongly alleviated through hypothermia treatment. Especially RGCs, which are affected in glaucoma disease, were protected due to a reduced apoptosis rate through hypothermia.

Destructive Effect of Intravitreal Heat Shock Protein 27 Application on Retinal Ganglion Cells and Neurofilament.

Heat shock protein 27 (HSP27) is commonly involved in cellular stress. Increased levels of HSP27 as well as autoantibodies against this protein were previously detected in glaucoma patients. Moreover, systemic immunization with HSP27 induced glaucoma-like damage in rodents. Now, for the first time, the direct effects of an intravitreal HSP27 application were investigated. For this reason, HSP27 or phosphate buffered saline (PBS, controls) was applied intravitreally in rats (n = 12/group). The intraocular pressure (IOP) as well as the electroretinogram recordings were comparable in HSP27 and control eyes 21 days after the injection. However, significantly fewer retinal ganglion cells (RGCs) and amacrine cells were observed in the HSP27 group via immunohistochemistry and western blot analysis. The number of bipolar cells, on the other hand, was similar in both groups. Interestingly, a stronger neurofilament degeneration was observed in HSP27 optic nerves, while no differences were noted regarding the myelination state. In summary, intravitreal HSP27 injection led to an IOP-independent glaucoma-like damage. A degeneration of RGCs as well as their axons and amacrine cells was noted. This suggests that high levels of extracellular HSP27 could have a direct damaging effect on RGCs.

Intravitreal S100B Injection Triggers a Time-Dependent Microglia Response in a Pro-Inflammatory Manner in Retina and Optic Nerve.

S100B is a glial protein, which belongs to calcium-binding protein family. Alterations of S100B level were noted in various neurodegenerative diseases. In a new glaucoma-like animal model S100B was injected intravitreally, which led to neuronal degeneration in retina and optic nerve. The pathological mechanisms are still unknown. Therefore, S100B protein was intravitreally injected in rats. At days 14 and 21, retina, optic nerve, serum, and aqueous humor were investigated. S100B injection led to an increase of retinal NF-κB at day 14. Furthermore, higher IL-1ß levels in retina, serum, and aqueous humor were measured. A co-localization of microglia and IL-1ß was noted, which correlated with an increased microglia response in retina and optic nerve at day 14. At the same point in time, more apoptotic RGCs and a decline in RGC numbers were observed. At 21 days, this damage was still present, but no signal pathway activations were detectable anymore. Interestingly, macroglia were not affected at any point in time. We conclude that S100B activated the NF-κB signal pathway, which then regulated IL-1ß production and release from microglia. A positive feedback loop of IL-1ß likely stimulates microglia in a pro-inflammatory manner. These microglia probably induce apoptotic damage in retina and optic nerve. Meanwhile, the injected S100B protein was naturally degraded, which explains the resting state of the pro-inflammatory signal pathways with constant damage later on. The inhibition of S100B release or microglia response could potentially decrease the damage in degenerative diseases, like glaucoma.

Diminished apoptosis in hypoxic porcine retina explant cultures through hypothermia.

Simulation of hypoxic processes in vitro can be achieved through cobalt chloride (CoCl2), which induces strong neurodegeneration. Hypoxia plays an important role in the progression of several retinal diseases. Thus, we investigated whether hypoxia can be reduced by hypothermia. Porcine retinal explants were cultivated for four and eight days and hypoxia was mimicked by adding 300 µM CoCl2 from day one to day three. Hypothermia treatment (30 °C) was applied simultaneously. Retinal ganglion, bipolar and amacrine cells, as well as microglia were evaluated via immunohistological and western blot analysis. Furthermore, quantitative real-time PCR was performed to analyze cellular stress and apoptosis. In addition, the expression of specific marker for the previously described cell types were investigated. A reduction of ROS and stress markers HSP70, iNOS, HIF-1α was achieved via hypothermia. In accordance, an inhibition of apoptotic proteins (caspase 3, caspase 8) and the cell cycle arrest gene p21 was found in hypothermia treated retinae. Furthermore, neurons of the inner retina were protected by hypothermia. In this study, we demonstrate that hypothermia lowers hypoxic processes and cellular stress. Additionally, hypothermia inhibits apoptosis and protects neurons. Hence, this seems to be a promising treatment for retinal neurodegeneration.

Intravitreal S100B Injection Leads to Progressive Glaucoma Like Damage in Retina and Optic Nerve.

The glial protein S100B, which belongs to a calcium binding protein family, is up-regulated in neurological diseases, like multiple sclerosis or glaucoma. In previous studies, S100B immunization led to retinal ganglion cell (RGC) loss in an experimental autoimmune glaucoma (EAG) model. Now, the direct degenerative impact of S100B on the retina and optic nerve was evaluated. Therefore, 2 µl of S100B was intravitreally injected in two concentrations (0.2 and 0.5 µg/µl). At day 3, 14 and 21, retinal neurons, such as RGCs, amacrine and bipolar cells, as well as apoptotic mechanisms were analyzed. Furthermore, neurofilaments, myelin fibers and axons of optic nerves were evaluated. In addition, retinal function and immunoglobulin G (IgG) level in the serum were measured. At day 3, RGCs were unaffected in the S100B groups, when compared to the PBS group. Later, at days 14 and 21, the RGC number as well as the ß-III tubulin protein level was reduced in the S100B groups. Only at day 14, active apoptotic mechanisms were noted. The number of amacrine cells was first affected at day 21, while the bipolar cell amount remained comparable to the PBS group. Also, the optic nerve neurofilament structure was damaged from day 3 on. At day 14, numerous swollen axons were observed. The intraocular injection of S100B is a new model for a glaucoma like degeneration. Although the application site was the eye, the optic nerve degenerated first, already at day 3. From day 14 on, retinal damage and loss of function was noted. The RGCs in the middle part of the retina were first affected. At day 21, the damage expanded and RGCs had degenerated in all areas of the retina as well as amacrine cells. Furthermore, elevated IgG levels in the serum were measured at day 21, which could be a sign of a late and S100B independet immune response. In summary, S100B had a direct destroying impact on the axons of the optic nerve. The damage of the retinal cell bodies seems to be a consequence of this axon loss.

Interaction of complement system and microglia activation in retina and optic nerve in a NMDA damage model.

It is known that intravitreally injected N-methyl-d-aspartate (NMDA) leads to fast retina and optic nerve degeneration and can directly activate microglia. Here, we analyzed the relevance for microglia related degenerating factors, the proteins of the complement system, at a late stage in the NMDA damage model. Therefore, different doses of NMDA (0 (PBS), 20, 40, 80 nmol) were intravitreally injected in rat eyes. Proliferative and activated microglia/macrophages (MG/Mϕ) were found in retina and optic nerve 2 weeks after NMDA injection. All three complement pathway proteins were activated in retinas after 40 and 80 nmol NMDA treatment. 80 nmol NMDA injection also lead to more numerous depositions of complement factors C3 and membrane attack complex (MAC) in retina and MAC in optic nerve. Additionally, more MAC+ depositions were detected in optic nerves of the 40 nmol NMDA group. In this NMDA model, the retina is first affected followed by optic nerve damage. However, we found initiating complement processes in the retina, while more deposits of the terminal complex were present 2 weeks after NMDA injection in the optic nerve. The complement system can be activated in waves and possibly a second wave is still on-going in the retina, while the first activation wave is in the final phase in the optic nerve. Only the damaged tissues showed microglia activation as well as proliferation and an increase of complement proteins. Interestingly, the microglia/macrophages (MG/Mϕ) in this model were closely connected with the inductors of the classical and lectin pathway, but not with the alternative pathway. However, all three initiating complement pathways were upregulated in the retina. The alternative pathway seems to be triggered by other mechanisms in this NMDA model. Our study showed an ongoing interaction of microglia and complement proteins in a late stage of a degenerative process.

Anti-inflammatory cytokine and angiogenic factors levels in vitreous samples of diabetic retinopathy patients.

Evaluation of cytokines in patients with diabetic retinopathy (DR) is important for the identification of future additive or alternative treatment options. Therefore, vitreous samples were obtained from patients with DR and patients with macular hole or macular pucker (control group) during 23-gauge-vitrectomy (n = 17/group). The levels of three pro-inflammatory (IL-1ß, IL-6, IFN-γ) and pleiotropic cytokines (IL-2, IL-4, IL-13) as well as VEGF, VEGF-A, and PGF were measured using an enzyme linked immunosorbent assay (ELISA). IL-1ß (p = 0.02) and IFN-γ (p = 0.04), two of the three tested pro-inflammatory cytokines, were elevated in the DR patients, while IL-6 (p = 0.51) level was comparable in both groups. Moreover, in DR samples, a trend towards an IL-13 down-regulation (p = 0.36) was observable. The IL-2 (p = 0.62) and IL-4 (p = 0.78) levels were comparable in both groups. All analyzed angiogenetic factors were up-regulated in DR patients (VEGF: p<0.001; VEGF-A: p = 0.002; PGF: p<0.001). The up-regulation of angiogenetic factors underline their importance in DR development. However, the interaction of the other cytokines showed an interesting pattern. Pro-inflammatory cytokines were also up-regulated, which could be evidence for inflammation processes in the diabetic retina. Furthermore, it seems that a counter response of immunomodulatory cytokines is in an initial process, but not strong enough to regulate the processes. Therefore, to support these anti-inflammatory mechanisms might be additive treatment option in the future.

HSP27 immunization reinforces AII amacrine cell and synapse damage induced by S100 in an autoimmune glaucoma model.

Previous studies have revealed a loss of retinal ganglion cells (RGCs) and optic nerve fibers after immunization with the S100B protein. Addition of heat shock protein 27 (HSP27) also leads to a decrease of RGCs. Our present aim has been to analyze various retinal cell types after immunization with S100B or S100B + HSP27 (S100 + HSP). After 28 days, retinas were processed for immunohistology and Western blot. RGCs, immunostained for NeuN, were significantly decreased in the S100 and the S100 + HSP groups. Significantly fewer ChAT+ cells were noted in both groups, whereas parvalbumin+ cells were only affected in the S100 + HSP group. Western blot results also revealed fewer ChAT signals in both immunized groups. No changes were noted with regard to PKCα+ rod bipolar cells, whereas a significant loss of recoverin+ cone bipolar cells was observed in both groups via immunohistology and Western blot. The presynaptic marker Bassoon and the postsynaptic marker PSD95 were significantly reduced in the S100 + HSP group. Opsin+ and rhodopsin+ photoreceptors revealed no changes in either group. Thus, the inner retinal layers are affected by immunization. However, the combination of S100 and HSP27 has a stronger additive effect on the retinal synapses and AII amacrine cells.

Concentration-Dependent Inner Retina Layer Damage and Optic Nerve Degeneration in a NMDA Model.

The intravitreal injection of N-methyl-D-aspartate (NMDA), a glutamate analogue, is an established model for fast retinal ganglion cell (RGC) degeneration. Yet, NMDA does not cause specific RGC damage. Now, the effects on the whole retina were analyzed. Additionally, the related effects for the structure and apoptotic levels of the optic nerve were investigated. Therefore, different NMDA concentrations were intravitreally injected in rats (20, 40, or 80 nmol NMDA or PBS). At days 3 and 14, Brn-3a+ RGCs were degenerated. A damage of calretinin+ amacrine cells was also recognized at day 14. Only a slight damage was observed in regard to PKCα+ bipolar cells, while rhodopsin+ photoreceptors remained intact. A long-lasting retinal microglia response was observed from day 3 up to day 14. Furthermore, a partial degeneration of the optic nerve was noted. At day 3, the SMI-32+ neurofilaments were just slightly affected, whereas the neurofilament structure was further degenerated at day 14. However, the luxol fast blue (LFB)-stained myelin structure remained intact from day 3 up to day 14. Interestingly, apoptotic mechanisms, like FasL and Fas co-localization as well as caspase 3 activation, were restricted to the optic nerve of the highest NMDA group at this late stage of degeneration. The degeneration of the optic nerve is probably only a side effect of neuronal degeneration of the inner retinal layers. The intact myelin structure might form a barrier against the direct influence of NMDA. In conclusion, this model is very suitable to test therapeutic agents, but it is important to analyze all inner retina layers and the optic nerve to determine their efficacy in this model more precisely.

A novel porcine ex vivo retina culture model for oxidative stress induced by H2O2.

Oxidative stress is a key player in many ophthalmic diseases. However, the role of oxidative stress in most degenerative processes is not yet known. Therefore, accurate and practical models are required to efficiently screen for therapeutics. Porcine eyes are closely related to the human eye, and can be obtained from the abattoir as a by-product of the food industry. Therefore, they offer excellent opportunities for the development of culture models with which to pre-screen potential therapies, while reducing the use of laboratory animals. To induce oxidative stress, organotypic cultures of porcine retina were treated with different doses of hydrogen peroxide (H2O2; 100, 300 and 500µM) for three hours. On days 3 and 8, the retinas were conserved for histological and Western blotting analyses and for evaluation of gene expression, which determined the number of retinal ganglion cells (RGCs), the activation state of glial cells, and the expression levels of several oxidative stress markers. H2O2 treatment led to a reduction in the number of RGCs and to an increase in apoptotic RGCs. In addition, a dose-dependent increase of microglia and an elevation of CD11b expression was observed. On day 3, a reduction of IL-1ß, and an increase of iNOS, as well as of HSP70 mRNA were found. On day 8, an increase in TNF-α and IL-1ß mRNA expression was detected. In conclusion, this ex vivo model offers an opportunity to study the molecular mechanisms underlying certain eye disorders and to test new therapeutic approaches to diminish the effects of oxidative stress.

Microglia response in retina and optic nerve in chronic experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a common rodent model for multiple sclerosis (MS). Yet, the long-term consequences for retina and optic nerve (ON) are unknown. C57BL/6 mice were immunized with an encephalitogenic peptide (MOG35-55) and the controls received the carriers or PBS. Clinical symptoms started at day 8, peaked at day 14, and were prevalent until day 60. They correlated with infiltration and demyelination of the ON. In MOG-immunized animals more microglia cells in the ONs and retinas were detected at day 60. Additionally, retinal ganglion cell (RGC) loss was combined with an increased macroglia response. At this late stage, an increased number of microglia was associated with axonal damage in the ON and in the retina with RGC loss. Whether glial activation contributes to repair mechanisms or adversely affects the number of RGCs is currently unclear.

Early remodelling of the extracellular matrix proteins tenascin-C and phosphacan in retina and optic nerve of an experimental autoimmune glaucoma model.

Glaucoma is characterized by the loss of retinal ganglion cells (RGCs) and optic nerve fibres. Previous studies noted fewer RGCs after immunization with ocular antigens at 28 days. It is known that changes in extracellular matrix (ECM) components conduct retina and optic nerve degeneration. Here, we focused on the remodelling of tenascin-C and phosphacan/receptor protein tyrosine phosphatase ß/ζ in an autoimmune glaucoma model. Rats were immunized with optic nerve homogenate (ONA) or S100B protein (S100). Controls received sodium chloride (Co). After 14 days, no changes in RGC number were noted in all groups. An increase in GFAP mRNA expression was observed in the S100 group, whereas no alterations were noted via immunohistochemistry in both groups. Extracellular matrix remodelling was analyzed after 3, 7, 14 and 28 days. Tenascin-C and 473HD immunoreactivity in retinae and optic nerves was unaltered in both immunized groups at 3 days. At 7 days, tenascin-C staining increased in both tissues in the ONA group. Also, in the optic nerves of the S100 group, an intense tenascin-C staining could be shown. In the retina, an increased tenascin-C expression was also observed in ONA animals via Western blot. 473HD immunoreactivity was elevated in the ONA group in both tissues and in the S100 optic nerves at 7 days. At 14 days, tenascin-C and 473HD immunoreactivity was up-regulated in the ONA retinae, whereas phosphacan expression was up-regulated in both groups. We conclude that remodelling of tenascin-C and phosphacan occurred shortly after immunization, already before RGC loss. We assume that both ECM molecules represent early indicators of neurodegeneration.