Glaucoma Animal Models beyond Chronic IOP Increase.

Glaucoma is a complex and multifactorial disease defined as the loss of retinal ganglion cells (RGCs) and their axons. Besides an elevated intraocular pressure (IOP), other mechanisms play a pivotal role in glaucoma onset and progression. For example, it is known that excitotoxicity, immunological alterations, ischemia, and oxidative stress contribute to the neurodegeneration in glaucoma disease. To study these effects and to discover novel therapeutic approaches, appropriate animal models are needed. In this review, we focus on various glaucoma animal models beyond an elevated IOP. We introduce genetically modified mice, e.g., the optineurin E50K knock-in or the glutamate aspartate transporter (GLAST)-deficient mouse. Excitotoxicity can be mimicked by injecting the glutamate analogue N-methyl-D-aspartate intravitreally, which leads to rapid RGC degeneration. To explore the contribution of the immune system, the experimental autoimmune glaucoma model can serve as a useful tool. Here, immunization with antigens led to glaucoma-like damage. The ischemic mechanism can be mimicked by inducing a high IOP for a certain amount of time in rodents, followed by reperfusion. Thereby, damage to the retina and the optic nerve occurs rapidly after ischemia/reperfusion. Lastly, we discuss the importance of optic nerve crush models as model systems for normal-tension glaucoma. In summary, various glaucoma models beyond IOP increase can be utilized.

Specific Biomarkers in the Aqueous Humour of Glaucoma Patients. / Spezifische Biomarker im Kammerwasser von Glaukompatienten.

Glaucoma, a multifactorial neurodegenerative disease, is the second most common cause of blindness. Since early diagnosis facilitates timely treatment, it is therefore essential to identify appropriate markers. In the future, so-called biomarkers could be helpful in early detection and follow-up. In glaucoma, these parameters could be obtained in the aqueous humour. Altered antibodies, proteins, microRNA (miRNA) and trace element levels have already been identified. This review provides insight into possible changes in the aqueous humour of patients with primary open-angle glaucoma (POAG), normal tension glaucoma (NTG) or pseudoexfoliation glaucoma (PEXG). Studies on antibody changes in POAG patients identified an upregulation of immune system associated antibodies such as heat shock protein (HSP) 27. HSP27 was also upregulated in PEXG patients but decreased in NTG. In POAG and PEXG samples, the levels of certain proteins, including interleukins and endothelin-1, were elevated. The vasoconstrictor endothelin-1 may play a role in regulating intraocular pressure. By contrast, proteins playing a role in the response to oxidative stress were downregulated. In NTG patients, proteins responsible for the elimination of toxic by-products from the respiratory chain were downregulated. In addition, the aqueous humour of POAG and PEXG patients contained several miRNAs that have been linked to tissue development, neurological disease and cellular organisation. Other miRNAs regulated in glaucoma play a role in extracellular matrix remodelling and thus may affect drainage resistance in the trabecular meshwork. It is also interesting to note that the aqueous humour of glaucoma patients showed changes in the levels of trace elements such as zinc and selenium. The elevated zinc levels could be responsible for the imbalance of intraocular matrix metalloproteinases and thus for increased intraocular pressure. All these studies demonstrate the complex changes in aqueous humour in glaucoma. Some of these biomarkers may be useful in the future for early diagnosis of the disease.

Heat Shock Protein 27 Injection Leads to Caspase Activation in the Visual Pathway and Retinal T-Cell Response.

Heat shock protein 27 (HSP27) is one of the small molecular chaperones and is involved in many cell mechanisms. Besides the known protective and helpful functions of intracellular HSP27, very little is known about the mode of action of extracellular HSP27. In a previous study, we showed that intravitreal injection of HSP27 led to neuronal damage in the retina and optic nerve after 21 days. However, it was not clear which degenerative signaling pathways were induced by the injection. For this reason, the pathological mechanisms of intravitreal HSP27 injection after 14 days were investigated. Histological and RT-qPCR analyses revealed an increase in endogenous HSP27 in the retina and an activation of components of the intrinsic and extrinsic apoptosis pathway. In addition, an increase in nucleus factor-kappa-light-chain-enhancer of activated B cells (NFκB), as well as of microglia/macrophages and T-cells could be observed. In the optic nerve, however, only an increased apoptosis rate was detectable. Therefore, the activation of caspases and the induction of an incipient immune response seem to be the main triggers for retinal degeneration in this intravitreal HSP27 model.

Activation of Apoptosis in a ßB1-CTGF Transgenic Mouse Model.

To reveal the pathomechanisms of glaucoma, a common cause of blindness, suitable animal models are needed. As previously shown, retinal ganglion cell and optic nerve degeneration occur in ßB1-CTGF mice. Here, we aimed to determine possible apoptotic mechanisms and degeneration of different retinal cells. Hence, retinae were processed for immunohistology (n = 5-9/group) and quantitative real-time PCR analysis (n = 5-7/group) in 5- and 10-week-old ßB1-CTGF and wildtype controls. We noted significantly more cleaved caspase 3+ cells in ßB1-CTGF retinae at 5 (p = 0.005) and 10 weeks (p = 0.02), and a significant upregulation of Casp3 and Bax/Bcl2 mRNA levels (p < 0.05). Furthermore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL+) cells were detected in transgenic mice at 5 (p = 0.03) and 10 weeks (p = 0.02). Neurofilament H staining (p = 0.01) as well as Nefh (p = 0.02) and Tubb3 (p = 0.009) mRNA levels were significantly decreased at 10 weeks. GABAergic synapse intensity was lower at 5 weeks, while no alterations were noted at 10 weeks. The glutamatergic synapse intensity was decreased at 5 (p = 0.007) and 10 weeks (p = 0.01). No changes were observed for bipolar cells, photoreceptors, and macroglia. We conclude that apoptotic processes and synapse loss precede neuronal death in this model. This slow progression rate makes the ßB1-CTGF mice a suitable model to study primary open-angle glaucoma.

Preservation of optic nerve structure by complement inhibition in experimental glaucoma.

Glaucoma is characterized by a progressive damage of the retina and the optic nerve. Despite a huge research interest, the exact pathomechanisms are still unknown. In the experimental autoimmune glaucoma model, rats develop glaucoma-like damage of the retina and the optic nerve after immunization with an optic nerve antigen homogenate (ONA). An early activation of the complement system, even before optic nerve degeneration, was reported in this model. Here, we investigated the effects of a monoclonal antibody against complement factor C5 on optic nerves. Rats were immunized with ONA and compared to controls. In one eye of some ONA animals, the antibody against C5 was intravitreally injected (15 µmol: ONA + C5-I or 25 µmol: ONA + C5-II) before immunization and then every 2 weeks. After 6 weeks, optic nerves were processed for histology (n = 6/group). These analyses demonstrated that the intravitreal therapy reduced the depositions of the membrane attack complex compared to ONA animals (ONA + C5-I: p = 0.005; ONA + C5-II: p = 0.002). Cellular infiltration was significantly reduced in the ONA + C5-I group (p = 0.003), but not in ONA + C5-II tissues (p = 0.41). Furthermore, SMI-32 staining revealed that neurofilament was preserved in both treatment groups compared to ONA optic nerves (both p = 0.002). A decreased amount of microglia was found in treated animals in comparison to the ONA group (ONA + C5-I: p = 0.03; ONA + C5-II: p = 0.009). We observed, for the first time, that a complement system inhibition could prevent optic nerve damage in an autoimmune glaucoma model. Therefore, complement inhibition could serve as a new therapeutic tool for glaucoma.

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.

[Current State of Primary Neuroprotection in Glaucoma]. / Derzeitiger Stand zur primären Neuroprotektion beim Glaukom.

In view of the aging members of our society, there will be an increase in severe visual impairment and blindness, also due to glaucoma, in the coming years. Therapy options are limited to treat occurring symptoms. Currently, only a deceleration of the pathogenesis progression, but no cure, is available. Therefore, it is necessary to develop new therapeutic strategies to treat glaucoma adequately and effectively, thus improving the quality of life of those affected. One possible approach seems to be primary neuroprotection, which acts independently of an intraocular pressure reduction. There are indications that components of the immune system play a role in the context of the disease or the loss of retinal ganglion cells. Thus, evidence of an involvement of heat shock proteins, the complement system, but also, for example, microglial cells, were found. To this end, therapeutic modulation of these factors seems to be an interesting new target for neuroprotection. Studies in animal models have shown that an inhibition of the complement system or microglia leads to a protection. Modulation of heat shock proteins may enhance their protective properties or inhibit their destroying function to prevent glaucoma damage. These neuroprotective substances could expand the treatment options of glaucoma patients in the future.

Loss of retinal ganglion cells in a new genetic mouse model for primary open-angle glaucoma.

Primary open-angle glaucoma (POAG) is one of the most common causes for blindness worldwide. Although an elevated intraocular pressure (IOP) is the main risk factor, the exact pathology remained indistinguishable. Therefore, it is necessary to have appropriate models to investigate these mechanisms. Here, we analysed a transgenic glaucoma mouse model (ßB1-CTGF) to elucidate new possible mechanisms of the disease. Therefore, IOP was measured in ßB1-CTGF and wildtype mice at 5, 10 and 15 weeks of age. At 5 and 10 weeks, the IOP in both groups were comparable (P > 0.05). After 15 weeks, a significant elevated IOP was measured in ßB1-CTGF mice (P < 0.001). At 15 weeks, electroretinogram measurements were performed and both the a- and b-wave amplitudes were significantly decreased in ßB1-CTGF retinae (both P < 0.01). Significantly fewer Brn-3a+ retinal ganglion cells (RGCs) were observed in the ßB1-CTGF group on flatmounts (P = 0.02), cross-sections (P < 0.001) and also via quantitative real-time PCR (P = 0.02). Additionally, significantly more cleaved caspase 3+ RGCs were seen in the ßB1-CTGF group (P = 0.002). Furthermore, a decrease in recoverin+ cells was observable in the ßB1-CTGF animals (P = 0.004). Accordingly, a significant down-regulation of Recoverin mRNA levels were noted (P < 0.001). Gfap expression, on the other hand, was higher in ßB1-CTGF retinae (P = 0.023). Additionally, more glutamine synthetase signal was noted (P = 0.04). Although no alterations were observed regarding photoreceptors via immunohistology, a significant decrease of Rhodopsin (P = 0.003) and Opsin mRNA (P = 0.03) was noted. We therefore assume that the ßB1-CTGF mouse could serve as an excellent model for better understanding the pathomechanisms in POAG.

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.

Role of Heat Shock Proteins in Glaucoma.

Glaucoma, one of the most common causes of blindness worldwide, is a multifactorial neurodegenerative disease characterized by damage of retinal ganglion cells and optic nerve degeneration. However, the exact mechanism leading to glaucoma is still not understood. Evidences suggest an immunological involvement in the pathogenesis. Among other immune responses, altered autoantibody patterns were found in glaucoma patients. Especially elevated antibody levels against heat shock proteins (HSPs), like HSP27 or HSP60, were identified. In an animal model, an immunization with these HSPs induced a pressure-independent retinal ganglion cell degeneration and axon loss, hence mimicking glaucoma-like damage. In addition, development of autoreactive antibodies, as well as a glia and T-cell activation, were described in these animals. Recently, we noted that intravitreal HSP27 injection likewise led to a degeneration of retinal ganglion cells and their axons. Therefore, HSP27 might have a direct damaging effect on retinal cells, and might play a key role in glaucoma.

Transfer of the Experimental Autoimmune Glaucoma Model from Rats to Mice-New Options to Study Glaucoma Disease.

Studies have suggested an involvement of the immune system in glaucoma. Hence, a rat experimental autoimmune glaucoma model (EAG) was developed to investigate the role of the immune response. Here, we transferred this model into mice. Either 0.8 mg/mL of the optic nerve antigen homogenate (ONA; ONA 0.8) or 1.0 mg/mL ONA (ONA 1.0) were injected in 129/Sv mice. Controls received sodium chloride. Before and 6 weeks after immunization, the intraocular pressure (IOP) was measured. At 6 weeks, retinal neurons, glia cells, and synapses were analyzed via immunohistology and quantitative real-time PCR (RT-qPCR). Additionally, optic nerves were examined. The IOP stayed in the normal physiological range throughout the study (p > 0.05). A significant reduction of retinal ganglion cells (RGCs) was noted in both immunized groups (p < 0.001). Remodeling of glutamatergic and GABAergic synapses was seen in ONA 1.0 retinas. Furthermore, both ONA groups revealed optic nerve degeneration and macrogliosis (all: p < 0.001). An increase of activated microglia was noted in ONA retinas and optic nerves (p < 0.05). Both ONA concentrations led to RGC loss and optic nerve degeneration. Therefore, the EAG model was successfully transferred from rats to mice. In further studies, transgenic knockout mice can be used to investigate the pathomechanisms of glaucoma more precisely.

Laquinimod protects the optic nerve and retina in an experimental autoimmune encephalomyelitis model.

BACKGROUND: The oral immunomodulatory agent laquinimod is currently evaluated for multiple sclerosis (MS) treatment. Phase II and III studies demonstrated a reduction of degenerative processes. In addition to anti-inflammatory effects, laquinimod might have neuroprotective properties, but its impact on the visual system, which is often affected by MS, is unknown. The aim of our study was to investigate potential protective effects of laquinimod on the optic nerve and retina in an experimental autoimmune encephalomyelitis (EAE) model. METHODS: We induced EAE in C57/BL6 mice via MOG35-55 immunization. Animals were divided into an untreated EAE group, three EAE groups receiving laquinimod (1, 5, or 25 mg/kg daily), starting the day post-immunization, and a non-immunized control group. Thirty days post-immunization, scotopic electroretinograms were carried out, and mice were sacrificed for histopathology (HE, LFB), immunohistochemistry (MBP, Iba1, Tmem119, F4/80, GFAP, vimentin, Brn-3a, cleaved caspase 3) of the optic nerve and retina, and retinal qRT-PCR analyses (Brn-3a, Iba1, Tmem119, AMWAP, CD68, GFAP). To evaluate the effect of a therapeutic approach, EAE animals were treated with 25 mg/kg laquinimod from day 16 when 60% of the animals had developed clinical signs of EAE. RESULTS: Laquinimod reduced neurological EAE symptoms and improved the neuronal electrical output of the inner nuclear layer compared to untreated EAE mice. Furthermore, cellular infiltration, especially recruited phagocytes, and demyelination in the optic nerve were reduced. Microglia were diminished in optic nerve and retina. Retinal macroglial signal was reduced under treatment, whereas in the optic nerve macroglia were not affected. Additionally, laquinimod preserved retinal ganglion cells and reduced apoptosis. A later treatment with laquinimod in a therapeutic approach led to a reduction of clinical signs and to an improved b-wave amplitude. However, no changes in cellular infiltration and demyelination of the optic nerves were observed. Also, the number of retinal ganglion cells remained unaltered. CONCLUSION: From our study, we deduce neuroprotective and anti-inflammatory effects of laquinimod on the optic nerve and retina in EAE mice, when animals were treated before any clinical signs were noted. Given the fact that the visual system is frequently affected by MS, the agent might be an interesting subject of further neuro-ophthalmic investigations.

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.

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.

In a novel autoimmune and high-pressure glaucoma model a complex immune response is induced.

Background: The neurodegenerative processes leading to glaucoma are complex. In addition to elevated intraocular pressure (IOP), an involvement of immunological mechanisms is most likely. In the new multifactorial glaucoma model, a combination of high IOP and optic nerve antigen (ONA) immunization leads to an enhanced loss of retinal ganglion cells accompanied by a higher number of microglia/macrophages in the inner retina. Here, we aimed to evaluate the immune response in this new model, especially the complement activation and the number of T-cells, for the first time. Further, the microglia/macrophage response was examined in more detail. Methods: Six-week-old wildtype (WT+ONA) and ßB1-connective tissue growth factor high-pressure mice (CTGF+ONA) were immunized with 1 mg ONA. A wildtype control (WT) and a CTGF group (CTGF) received NaCl instead. Six weeks after immunization, retinae from all four groups were processed for immunohistology, RT-qPCR, and flow cytometry, while serum was used for microarray analyses. Results: We noticed elevated numbers of C1q+ cells (classical complement pathway) in CTGF and CTGF+ONA retinae as well as an upregulation of C1qa, C1qb, and C1qc mRNA levels in these groups. While the complement C3 was only increased in CTGF and CTGF+ONA retinae, enhanced numbers of the terminal membrane attack complex were noted in all three glaucoma groups. Flow cytometry and RT-qPCR analyses revealed an enhancement of different microglia/macrophages markers, including CD11b, especially in CTGF and CTGF+ONA retinae. Interestingly, increased retinal mRNA as well as serum levels of the tumor necrosis factor α were found throughout the different glaucoma groups. Lastly, more T-cells could be observed in the ganglion cell layer of the new CTGF+ONA model. Conclusion: These results emphasize an involvement of the complement system, microglia/macrophages, and T-cells in glaucomatous disease. Moreover, in the new multifactorial glaucoma model, increased IOP in combination with autoimmune processes seem to enforce an additional T-cell response, leading to a more persistent pathology. Hence, this new model mimics the pathomechanisms occurring in human glaucoma more accurately and could therefore be a helpful tool to find new therapeutic approaches for patients in the future.

Immune response against ocular tissues after immunization with optic nerve antigens in a model of autoimmune glaucoma.

PURPOSE: In recent years, numerous studies have investigated the involvement of immunological mechanisms in glaucoma. Until now, it has not been determined whether the altered antibody pattern detected in patients is harmful to retinal ganglion cells (RGCs) or triggers disease formation in any way. In a model of experimental autoimmune glaucoma, RGC loss can be induced through immunization with certain ocular antigens. In the current study, the time course of the levels of autoreactivity against ocular tissues after immunization was examined. METHODS: Intraocular pressure was measured regularly. Ten weeks after immunization with an optic nerve homogenate antigen (ONA), the number of RGCs was determined. Immunoglobulin G levels in aqueous humor were measured via enzyme-linked immunosorbent assay at the same time point. Serum from different time points was used to analyze the possible occurrence of autoreactive antibodies against the retina or optic nerve in this autoimmune glaucoma model. Additionally, optic nerve and brain sections were evaluated for possible pathological findings. RESULTS: Intraocular pressure stayed within the normal range throughout this study. A continuous increase of autoreactive antibodies against the optic nerve and retina sections was observed. At 4, 6, and 10 weeks, antibody reactivity was significantly higher in ONA animals (p<0.01). Aqueous humor immunoglobulin G levels were also significantly higher in the ONA group (p=0.006). Ten weeks after immunization, significantly fewer RGCs were noted in the ONA group (p=0.00003). The optic nerves from ONA animals exhibited damaged axons. No pathological findings appeared in any brain sections. CONCLUSIONS: Our findings suggest that these modified antibodies play a substantial role in mechanisms leading to RGC death. The slow dissolution of RGCs observed in animals with autoimmune glaucoma is comparable to the slow progressive RGC loss in glaucoma patients, thus making this a useful model to develop neuroprotective therapies in the future.

HSP27 induced glaucomatous damage in mice of young and advanced age.

Introduction: Age-related diseases such as glaucoma, a leading cause of blindness, are having an upward trend due to an aging society. In glaucoma, some patients display altered antibody profiles and increased antibody titers, for example against heat shock protein 27 (HSP27). An intravitreal injection of HSP27 leads to glaucoma-like damage in rats. We now aimed to investigate if aged mice are more prone to this damage than younger ones. Methods: We intravitreally injected HSP27 into young (1-2 months) and aged (7-8 months) mice to compare glaucomatous damage. Respective age-matched controls received PBS. Not injected eyes served as naive controls. Results: Optical coherence tomography 4 weeks after injection showed no changes in retinal thickness in all groups at both ages. Cell counts and RT-qPCR revealed a significant reduction in RGC numbers in HSP27 mice at both ages. Comparing aged and young HSP27 mice, no differences in Rbpms and Pou4f1 (RGCs) expression was detected, while the Tubb3 expression (neuronal cells) was significantly upregulated in aged HSP27 animals. Neither microglia/macrophages nor (resident) microglia counts revealed significant differences in HSP27 mice at both ages. Nevertheless, increased relative Iba1 and Tmem119 expression was detected in young and aged HSP27 mice. Aged HSP27 mice displayed a significantly lower Iba1 expression than young ones, whereas Cd68 levels were upregulated. A larger GFAP+ area and an upregulation of GFAP expression in HSP27 animals of both ages indicated a macrogliosis. Also, elevated Il1b and Nos2 expression levels were observed in young and aged HSP27 mice. However, only Il1b levels were upregulated when comparing 7-8 months to 1-2 months old animals. A larger HSP25+ area was seen in aged HSP27 animals, while Hspb2 expression levels were downregulated in both HSP27 groups. The aged HSP27 group displayed an upregulated Hspb2 expression compared to young mice. Furthermore, a higher optic nerve degeneration score was noted in young and aged HSP27 groups. Discussion: These findings indicate that an intravitreal injection of HSP27 led to RGC loss accompanied by inflammation. Age-dependent effects (7-8 months vs. 1-2 months) were not very prominent. The results suggest a potential role of extracellular HSP27 in the development of glaucoma.

Attitudes towards sex workers: a nationwide cross-sectional survey among German healthcare providers.

Background: Worldwide, sex workers face stigmatization and discrimination, also within healthcare. Only few studies on healthcare providers' attitudes towards care of sex workers have been performed. This study assessed attitudes and knowledge of healthcare providers in Germany towards sex workers and their specific health risks. Methods: German healthcare professionals and medical students were invited to participate in a nationwide cross-sectional study in 2022. The online survey used a German translation of the "Attitudes towards Prostitutes and Prostitution Scale" by Levin and Peled for assessment of attitudes towards sex work and workers, together with prevalence estimates of common mental and physical disorders. Results: A total of 469 questionnaires were included into analysis. Older participants tended to regard sex work as less of a choice (p < 0.004) and sex workers as more victimized (p < 0.001). The frequency of professional contact to sex workers neither affected the perception of sex workers' status as victims vs. independent individuals, nor the perceived moral status. Moreover, healthcare professionals overestimated the prevalence of various disorders which was influenced by participants' attitudes towards sex workers. Discussion: A comparison to a recent Allensbach survey demonstrated similar attitudes of healthcare providers and the general population towards sex workers. Our results suggest that German healthcare professionals are not free of prejudices against sex workers, as has been shown for other marginalized groups in society. Instead, they seem to be influenced by personal opinion rather than by objective facts which they should have acquired during their professional education. Future interventions (e.g., better training regarding marginal societal groups) are necessary to encounter these issues in order to improve healthcare for sex workers.

Investigation of Inter- and Intra-Day Variability of Tear Fluid Regarding Flow Rate, Protein Concentration as well as Protein Composition.

Purpose: The purpose of this study was to present the determination of inter- and intra-day variations in tear flow rate, and tear fluid protein concentration, as well as protein composition regarding their impact for future biomarker studies. Methods: Tear fluid was collected noninvasively from 18 healthy subjects by performing Schirmer tests at 4 different time points repetitive in a period of 2 days. The tear flow rate on the Schirmer test strips was measured. Proteins were extracted from strips and quantified using amino acid analysis. Protein composition was analyzed by the strips data-independent (DIA) based mass spectrometry. To exclude any impairments to health, volunteers underwent a detailed neurological as well as an ophthalmological examination. Results: Whether tear fluid was collected from oculus sinister or oculus dexter did not affect the tear flow rate (P ≈ 0.63) or protein concentration (P ≈ 0.97) of individual subjects. Moreover, protein concentration was independent from the tear volume, so that a change in volume may only influence the total protein amount. When the examination days were compared, investigation of tear flow rate (P ≈ 0.001) and protein concentration (P ≈ 0.0003) indicated significant differences. Further, mass spectrometric analysis of tear fluid revealed 11 differentially regulated proteins when comparing both examination days. Conclusions: Our findings provide evidence of inter-day variation in tear flow rate, tear proteome concentration, and composition in healthy subjects, suggesting that inter-day variation needs to be taken into consideration in biomarker research of tear fluid. Identified proteins were assigned to functions in the immune response, oxidative and reducing processes, as well as mannose metabolism.