Background:Currently, the treatment of glaucoma is limited to reducing intraocular pressure since other involved pathomechanisms are not well understood yet. Evidence points to an immune-mediated component in disease development. For example, elevated antibody levels against heat shock protein 27 (HSP27) were detected in glaucoma patients. In mice, we previously noted glaucoma-like damage after an intravitreal HSP27 injection. Now, we aimed to investigate if intermittent fasting protects from this glaucomatous damage. Methods:CD1 mice were intravitreally injected with HSP27 into one eye. The contralateral eye served as a control. After injections, half of the animals received food ad libitum (no diet). The other half fasted, hence access to food was denied for 24 h at three days per week (diet). The animals were weighed weekly. Retinal thickness was analyzed via optical coherence tomography (OCT) after 4 weeks (n = 7 eyes/group). Via immunohistology, retinal ganglion cells (RGCs), apoptotic cells, macroglia, microglia/macrophages, tumor necrosis factor (TNFα), and interleukin (IL)-1β were analyzed (n = 6 eyes/group). Corresponding markers were examined with RT-qPCR (n = 4 samples/group). In addition, microarray assays were performed from serum samples from mice with diet or with no diet (n = 6 samples/group). Results:The weight and OCT measurements revealed no differences between the groups. HSP27 retinas had significantly lower RGC numbers as well as decreased Rbmps mRNA levels compared to controls, while HSP27+diet retinas displayed similar RGC counts as controls. No difference was observed in apoptotic markers. The macroglia+ area was increased in HSP27 tissue, while the HSP27+diet group showed no difference to controls. The number of microglia was not altered after HSP27 injection but was lower in HSP27+diet retinas. Tnfa and Il1b expression levels were downregulated in HSP27+diet samples compared to control as well as HSP27 tissue. Moreover, different pro-inflammatory cytokines, including IL-1β and IL-6, were lower in the serum of diet mice compared to no diet ones. Conclusion:Intravitreal injection of HSP27 resulted in RGC loss and was associated with gliosis. In contrast, intermittent fasting conferred neuroprotective effects, likely by modulating neuroinflammatory pathways, and hence protected RGCs from damage. These findings highlight intermittent fasting as a potential adjunctive therapeutic strategy for glaucoma management.

Neurodegenerative diseases, such as glaucoma or multiple sclerosis, are characterized by progressive neuronal loss involving diverse pathogenic mechanisms. The brain-derived neurotrophic factor (BDNF) has been implicated in neuroprotection and neural plasticity, yet its regulation and involvement in retinal neurodegenerative diseases remain largely unclear. In this study, we investigated the impact of BDNF deficiency in immune cells on retinal integrity. Using mice with a conditional BDNF knockout in microglia/macrophages and T-cells or selectively in microglia/macrophages, we analyzed retinal changes at 3 and 7 months of age, with wildtype mice as controls. BDNF-deficient mice exhibited early and progressive degeneration of retinal ganglion cells and photoreceptors, accompanied by pronounced astrogliosis, which was exacerbated in aged animals. In 7-month-old mice, adaptive changes in synapses could be documented, evidenced through enhanced expression of the vesicular acetylcholine transporter. These findings demonstrate that BDNF from immune cells plays a crucial role in maintaining retinal homeostasis and that its loss promotes retinal neurodegeneration. Targeting immune cell-derived BDNF may offer novel therapeutic strategies for retinal involvement in neurodegenerative diseases with implications for treatment of glaucoma or multiple sclerosis.

Besides an elevated intraocular pressure (IOP), advanced age is one of the most crucial risk factors for developing glaucoma. βB1-Connective Tissue Growth Factor (βB1-CTGF) high-pressure glaucoma mice were used in this study to assess whether glaucoma mice display more inflammatory and aging processes than age-matched controls. Therefore, 20-month-old βB1-CTGF and corresponding wildtype (WT) controls were examined. After IOP measurements, retinas were processed for (immuno-)histological and quantitative real-time PCR analyses. A significantly higher IOP and diminished retinal ganglion cell numbers were noted in βB1-CTGF mice compared to WT. An enhanced macrogliosis as well as an increased number of microglia/macrophages and microglia was detected in retinas of old glaucoma mice. Interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and transforming growth factor-β2 were upregulated, suggesting an ongoing inflammation. Moreover, βB1-CTGF retinas displayed an increased senescence-associated β-galactosidase staining accompanied by a downregulation of Lmnb1 (laminin-B1) mRNA levels. Our results provide a deeper insight into the association between inflammation and high-pressure glaucoma and thus might help to develop new therapy strategies.

Microglia play a critical role in central nervous system (CNS) pathologies including multiple sclerosis (MS), and their modulation offers therapeutic potential especially during progressive disease courses. Using cell culture and experimental autoimmune encephalomyelitis (EAE) models, we investigated microglial dynamics during depletion and repopulation (MGrepo) and their modulation using siponimod (sipo), an established CNS penetrating MS medication. Repopulating microglia exhibited a transient reactive state (CD86, MHC-II, Il1b, Tnf) . Sipo modulated microglia populations, increasing CD163+, CD206+, and CX3CR1+ while reducing CD86+MHC-II+ cells accompanied by a reduction of neuronal damage. Proteomic spinal cord analysis revealed protein expression alterations by MGrepo and sipo linked to inflammation, myelination, and neuronal structural organization, supported by RNA sequencing of the spinal cord. The neuroinflammation attenuating role of sipo could be linked to cell maintenance and myelin formation associated processes. These findings highlight the capacity of pharmacological interventions to modulate microglia, offering new insights into therapeutic strategies targeting microglial activity in neuroinflammatory diseases. ### Competing Interest Statement The authors declare the following potential conflicts of interest, all not related to the content of this manuscript: NH, SaF, LR, ACG, HHH, SO, KK, RH, AK, HM, CPS, SR, KFW, SCJ, JRP, BE, MVM, SR, ME,VB, GT, OG, KMA, IS have nothing to disclose. VWY is funded by research grants from MS Canada, the Canadian Institutes of Health Research, USA Department of Defense Multiple Sclerosis Research Program, Genentech and Novartis. He has received speaker honoraria from Biogen, EMD Serono, Novartis, Roche, Sanofi-Genzyme and Teva Canada. He is the recipient of unrestricted educational grants from Biogen, EMD Serono, Novartis, Roche, Sanofi-Genzyme and Teva Canada to support educational activities of the Alberta MS Network, which he directs. RG serves on scientific advisory boards for Teva Pharmaceutical Industries Ltd., Biogen, Bayer Schering Pharma, and Novartis; has received speaker honoraria from Biogen, Teva Pharmaceutical Industries Ltd., Bayer Schering Pharma, Merck and Novartis; serves as editor for Therapeutic Advances in Neurological Diseases and on the editorial boards of Experimental Neurology and the Journal of Neuroimmunology; and receives research support from Teva Pharmaceutical Industries Ltd., Biogen Idec, Bayer Schering Pharma, Genzyme, Merck Serono, and Novartis. * BP : biological processes CNS : central nervous system DEGs : differentially expressed genes DT : diphtheria toxin DTR : diphtheria toxin receptor EAE : experimental autoimmune encephalomyelitis FC : fold change GO Term : gene ontology term of biological processes H&E : hematoxylin and eosin ICC : immunocytochemistry iLPS : interferon-gamma and lipopolysaccharide LFB : luxol fast blue MGrepo : microglia depletion and repopulation MS : Multiple sclerosis pMS : progressive forms of MS PPMS : Primary progressive multiple sclerosis PCA : principal component analysis PI : propidium iodide PMA/Iono : phorbol 12-myristate 13-acetate/ionomycin PNS : peripheral nervous system pwMS : people living with MS qRT.PCR : quantitative reverse transcriptase polymerase chain reaction RRMS : Relapsing-remitting multiple sclerosis SEM : Standard error of the mean Sipo : siponimod SPMS : Secondary progressive multiple sclerosis S1PR : sphingosine 1 phosphate receptor TAM : tamoxifen Tc cells : Cytotoxic T cells Tcons : conventional T cells Th cells : T helper cells Treg cells : Regulatory T cells Medical faculty Ruhr-University Bochum, N/A Novartis (Germany), https://ror.org/0013shd50, N/A

With aging, senescence-related diseases are increasing in prevalence. The senescence of cells in the central nervous system has been linked with the development of neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease. These changes are not limited to the brain as many eye diseases, such as cataract, diabetic retinopathy, age-related macular degeneration, and glaucoma, are also age-related. Among them, glaucoma is one of the leading causes of irreversible blindness with a multifactorial neurodegenerative nature. Besides an elevated intraocular pressure, an increased age is one of the main risk factors for this disease. Hence, in this review, we will discuss age-related changes in the context of eye disease, with a specific focus on glaucoma. Several general aging mechanisms were put forward in different eye diseases. This includes dysregulated nutrient sensing, cellular senescence, stem cell exhaustion, altered intercellular communication, genomic instability, telomere shortening, epigenetic alteration, loss of proteostasis, compromised autophagy, and mitochondrial dysfunction. In glaucoma, aging is a main risk factor for the development. This is triggered by oxidative, metabolic, immunological, and biomechanical stressors with many cross-talks. Oxidative stress, for example, can also trigger apoptotic cell death through mitochondrial damage, hypoxia, inflammation, and endothelial dysregulation. Also, with advanced age, alterations in extracellular matrix composition and structure are becoming important biomechanical contributing factors to the pathology of glaucoma. All mentioned mechanisms triggered by aging processes are generally accepted as contributing factors in the development of glaucoma in the aged eye. A better understanding of these will help to find novel therapeutic approaches for glaucoma patients in the future.

BackgroundThe 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.MethodsSix-week-old wildtype (WT+ONA) and βB1-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.ResultsWe 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.ConclusionThese 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.

IntroductionOne of the most common causes of vision loss in the elderly population worldwide is age-related macular degeneration (AMD). Subsequently, the number of people affected by AMD is estimated to reach approximately 288 million by the year 2040. The aim of this study was to develop an ex vivo model that simulates various aspects of the complex AMD pathogenesis.MethodsFor this purpose, primary porcine retinal pigment epithelial cells (ppRPE) were isolated and cultured. One group was exposed to medium containing sodium iodate (NaIO3) to induce degeneration. The others were exposed to different supplemented media, such as bovine serum albumin (BSA), homogenized porcine retinas (HPR), or rod outer segments (ROOS) for eight days to promote retinal deposits. Then, these ppRPE cells were cocultured with porcine neuroretina explants for another eight days. To assess the viability of ppRPE cells, live/dead assay was performed at the end of the study. The positive RPE65 and ZO1 area was evaluated by immunocytochemistry and the expression of RLBP1, RPE65, and TJP1 was analyzed by RT-qPCR. Additionally, drusen (APOE), inflammation (ITGAM, IL6, IL8, NLRP3, TNF), oxidative stress (NFE2L2, SOD1, SOD2), and hypoxia (HIF1A) markers were investigated. The concentration of the inflammatory cytokines IL-6 and IL-8 was determined in medium supernatants from day 16 and 24 via ELISA.ResultsLive/dead assay suggests that especially exposure to NaIO3 and HPR induced damage to ppRPE cells, leading in a significant ppRPE cell loss. All supplemented media resulted in decreased RPE-characteristic markers (RPE65; ZO-1) and gene expression like RLBP1 and RPE65 in the cultured ppRPE cells. Besides, some inflammatory, oxidative as well as hypoxic stress markers were altered in ppRPE cells cultivated with NaIO3. The application of HPR induced an enhanced APOE expression. Pre-exposure of the ppRPE cells led to a diminished number of cones in all supplemented media groups compared to controls.DiscussionOverall, this novel coculture model represents an interesting initial approach to incorporating deposits into coculture to mimic AMD pathogenesis. Nevertheless, the effects of the media used need to be investigated in further studies.

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.

BackgroundSexual health is an important aspect of human well-being. In terms of sexual health and healthcare, sex workers might need more specialized care than others, given their higher risk for both discrimination and various sexually transmitted diseases. However, little is known about the quality of healthcare professionals' training regarding sexual health and healthcare of sex workers in Germany.MethodsIn an online survey, 130 physicians, 63 psychotherapists, and 154 medical students reported their perceived quality of training regarding sexual health problems in all their patients and regarding sex workers' health issues specifically.ResultsA substantial share of respondents reported to have experienced inadequate training regarding both sexual health problems in general and specific issues concerning sex workers. However, most respondents reported feeling rather comfortable when dealing with these topics. There was a positive correlation between feeling comfortable when treating sexual health problems/ sex workers and the perceived training on these topics.ConclusionsThe results of this study indicate that sexual health issues and sex workers' specific healthcare requirements are not sufficiently addressed in the curricula of German healthcare professionals. Future adaptations of these curricula might be necessary.

>Glaucoma is a group of eye diseases characterized by progressive loss of retinal ganglion cells(RGCs) and optic nerve degeneration.During this process,the visual field is reduced,and blindness may ultimately occur.Worldwide,glaucoma is the second leading cause of blindness,with about 80 million people affected.Glaucoma is a multifactorial disease and due to its complexity,

>In a further aging society, excellent eyesight is an integral part of overall well-being and quality of life. Preserving good vision is crucial to maintaining mobility, independence, and mental health. There can be several reasons for visual impairment in elderly people,

PURPOSE. The purpose of this study was to present the determination of inter-and intraday 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 approximate to 0.63) or protein concentration (P approximate to 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 approximate to 0.001) and protein concentration (P approximate to 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.