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.

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.

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.

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.

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.

Heat Shock Protein Upregulation Supplemental to Complex mRNA Alterations in Autoimmune Glaucoma.

Glaucomatous optic neuropathy is a common cause for blindness. An elevated intraocular pressure is the main risk factor, but also a contribution of the immune system seems likely. In the experimental autoimmune glaucoma model used here, systemic immunization with an optic nerve homogenate antigen (ONA) leads to retinal ganglion cell (RGC) and optic nerve degeneration. We processed retinae for quantitative real-time PCR and immunohistology 28 days after immunization. Furthermore, we performed mRNA profiling in this model for the first time. We detected a significant RGC loss in the ONA retinae. This was accompanied by an upregulation of mRNA expression of genes belonging to the heat shock protein family. Furthermore, mRNA expression levels of the genes of the immune system, such as C1qa, C1qb, Il18, and Nfkb1, were upregulated in ONA animals. After laser microdissection, inner retinal layers were used for mRNA microarrays. Nine of these probes were significantly upregulated in ONA animals (p < 0.05), including Hba-a1 and Cxcl10, while fifteen probes were significantly downregulated in ONA animals (p < 0.05), such as Gdf15 and Wwox. Taken together, these findings provide further insights into the pivotal role of the immune response in glaucomatous optic neuropathy and could help to identify novel diagnostic or therapeutic strategies.

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.

Enhanced glaucomatous damage accompanied by glial response in a new multifactorial mouse model.

Introduction: Glaucoma is a complex, multifactorial neurodegenerative disease, which can lead to blindness if left untreated. It seems that, among others, immune processes, elevated intraocular pressure (IOP), or a combination of these factors are responsible for glaucomatous damage. Here, we combined two glaucoma models to examine if a combination of risk factors (IOP and immune response) results in a more severe damage of retinal ganglion cells (RGCs) and the optic nerves as well as an additional glia activation. Methods: Six-week-old wildtype (WT+ONA) and ßB1-Connective Tissue Growth Factor (CTGF) mice (CTGF+ONA) were immunized with 1 mg ONA (optic nerve antigen). A WT and a CTGF control group (CTGF) received sodium chloride instead. IOP was measured before and every two weeks after immunization. After six weeks, electroretinogram (ERG) measurements were performed. Then, retinae and optic nerves were processed for (immuno-) histology. Further, mRNA levels of corresponding genes in optic nerve and retina were analyzed via RT-qPCR. Results: Six weeks after immunization, the IOP in CTGF and CTGF+ONA mice was increased. The optic nerve of CTGF+ONA animals displayed the most severe cell inflammation, demyelination, and macroglia activation. Fewer numbers of oligodendrocytes were only observed in WT+ONA optic nerves, while more apoptotic cells triggered by the extrinsic pathway could be revealed in all three glaucoma groups. The number of microglia/macrophages was not altered within the optic nerves of all groups. The loss of neuronal cells, especially RGCs was most pronounced in CTGF+ONA retinae in the central part and this was accompanied by an enhanced activation of microglia/macrophages. Also, Müller cell activation could be noted in CTGF and CTGF+ONA retinae. Discussion: In this new model, an additive degeneration could be noted in optic nerves as well as in the number of RGCs. These results suggest a potential additive role of high IOP and immune factors in glaucoma development, which will aid for understanding this multifactorial disease more precisely in the future.

Cytokine and Complement Response in the Glaucomatous ßB1-CTGF Mouse Model.

Glaucoma is a complex neurodegenerative disease leading to a loss of retinal ganglion cells (RGCs) and optic nerve axons. An activation of the complement system seems to contribute to cell loss in this disease. Hence, we investigated a possible initiation of the complement system and the cytokine response in the ßB1-CTGF glaucoma model. In these mice, intraocular pressure is elevated, which is the main glaucoma risk factor in patients, and RGC loss occurs at 15 weeks of age. Therefore, quantitative real-time PCR and immunohistological experiments were performed in 5-, 10-, and 15-week-old ßB1-CTGF animals and their corresponding wildtypes (WT) to analyze the expression of several complement system factors. We could show that mRNA levels of the terminal complement pathway components C3 and C5 (Hc) were upregulated at 10 weeks. In accordance, more C3+ and membrane attack complex+ cells were observed in transgenic retinae. Further, the C5a receptor anaphylatoxin receptor (C5ar) and the complement component C5a receptor 1 (C5ar1; CD88) mRNA levels were upregulated in 10- and 15-week-old ßB1-CTGF mice. Interestingly, all three activation routes of the complement system were elevated in ßB1-CTGF mice at some age. Especially C1q, as a marker of the classical pathway, was significantly increased at all investigated ages. Furthermore, mRNA expression levels of interferon-γ (Infg) were upregulated at 5 weeks, while Cxcl1 and Cxcl2 mRNA levels were upregulated at 10 and 15 weeks. The mRNA levels of the chemokines Cxcl10 were increased at all ages in ßB1-CTGF mice. These results lead to the assumption that in these transgenic mice, a complement activation mainly through the classical pathway as well as a cytokine response plays a major role in cell death.

Epidermal Growth Factor Is Increased in Conjunctival Malignant Melanoma.

BACKGROUND/AIM: Conjunctival malignant melanoma (CMM) is a rare, but very aggressive tumor with a high metastasis rate. Not much is known about the CMM metastasis mechanisms. So far, epidermal growth factor (EGF) and its receptor (EGF-R) as well as macrophages and matrix metalloproteinase 9 (MMP-9) have been reported to lead to metastasis by epithelial-mesenchymal-transition and tumor migration in different solid tumors. Therefore, we evaluated whether EGF and EGF-R, CD68 and MMP-9 are altered in CMM samples in comparison to conjunctival nevi and healthy conjunctiva. PATIENTS AND METHODS: EGF, EGF-R, the macrophage marker CD68 and MMP-9 expression were analyzed in human conjunctival melanoma (CMM, n=16), human conjunctival nevi (n=13) and disease-free human conjunctiva (controls, n=14) by immunohistology. Staining of each sample was evaluated using a standardized score ranging from negative (0) to triple positive (3). The groups were then compared by ANOVA, followed by Tukey's post-hoc test. RESULTS: A statistically significant increase of EGF was seen in CMM samples in comparison to conjunctival nevi (p=0.03). In contrast, no statistically significant differences in EGF-R expression were noted between the three groups. A statistically significant increase of CD68 was only seen in conjunctival nevi compared to controls (p=0.04). MMP-9 expression was similar in all groups. CONCLUSION: In CMM, the study data demonstrated an up-regulation of EGF in comparison to conjunctival nevi. Hence, EGF might promote proliferation of CMM cells and induce the epithelial-mesenchymal transition. Therefore, our data suggest that an interplay between EGF and CMM might have a critical role in the developing CMM tumors and metastasis.

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.

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.