Protection against Oxidative Stress by Coenzyme Q10 in a Porcine Retinal Degeneration Model.

Oxidative stress plays an important role in neurodegenerative diseases, including glaucoma. Therefore, we analyzed if the antioxidant coenzyme Q10 (CoQ10), which is also commercially available, can prevent retinal degeneration induced by hydrogen peroxide (H2O2) in a porcine organ culture model. Retinal explants were cultivated for eight days, and H2O2 (500 µM, 3 h) induced the oxidative damage. CoQ10 therapy was applied (700 µM, 48 h). Retinal ganglion cells (RGCs) and microglia were examined immunohistologically in all groups (control, H2O2, H2O2 + CoQ10). Cellular, oxidative, and inflammatory genes were quantified via RT-qPCR. Strong RGC loss was observed with H2O2 (p ≤ 0.001). CoQ10 elicited RGC protection compared to the damaged group at a histological (p ≤ 0.001) and mRNA level. We detected more microglia cells with H2O2, but CoQ10 reduced this effect (p = 0.004). Cellular protection genes (NRF2) against oxidative stress were stimulated by CoQ10 (p ≤ 0.001). Furthermore, mitochondrial oxidative stress (SOD2) increased through H2O2 (p = 0.038), and CoQ10 reduced it to control level. Our novel results indicate neuroprotection via CoQ10 in porcine retina organ cultures. In particular, CoQ10 appears to protect RGCs by potentially inhibiting apoptosis-related pathways, activating intracellular protection and reducing mitochondrial stress.

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.

Changes of Subjective Symptoms and Tear Film Biomarkers Following Lenticule Extraction.

PURPOSE: To investigate the influence of lenticule extraction on subjective symptoms and objective biomarkers of dry eye and to clarify relationships between markers and find indicators for subjective symptoms after lenticule extraction. METHODS: Right eyes of myopic patients undergoing lenticule extraction surgery (n = 35) were examined preoperatively and 5 and 90 days postoperatively using established clinical dry eye examination methods (tear film break-up time [BUT], Schirmer test, lissamine green and fluorescein staining, and Ocular Surface Disease Index (OSDI) questionnaire). A patient subset was also examined after 1 year (n = 14). Tear samples were eluted from Schirmer strips and then measured with enzyme-linked immunosorbent assays (calcitonin gene-related peptide (CGRP), interleukin-1ß, IL-6, IL-8, matrix metallopeptidase 9 [MMP-9], nerve growth factor, and tumor necrosis factor-α). Postoperatively, unpreserved ofloxacin and dexamethasone eye drops were given (four times a day for 10 days). RESULTS: BUT decreased at days 5 (P = .023) and 90 (P = .025). Lissamine green staining increased at day 90 (P = .036). OSDI values increased at day 5 (total values, vision-related function, ocular symptoms, all P < .001, but not environmental triggers) and at day 90 (vision-related function, P = .017). A downregulation of CGRP (P = .006) and MMP-9 levels (P = .042) was observed on day 5 compared to day 90. CONCLUSIONS: Due to incongruity of patient symptoms, clinical signs, and tear protein changes, no predictive indicator was found, but some patients reported increased discomfort. Changes after lenticule extraction are not exclusively due to dry eye. [J Refract Surg. 2023;39(9):597-604.].

Etiology of Idiopathic Macular Holes in the Light of Estrogen Hormone.

The aim of this review was to identify a new potential explanation for the development of macular holes in relation to the female sex and to explain the possible underlying pathways. This approach was based on the evaluation of anatomical, physiological, and morphological analyses currently available in the literature. The findings showed that estrogen exerts a protective effect on the neuroretina and may influence Müller and cone cells. Both cell types are responsible for the building of the fovea structure. However, this protection may be lost due to the sudden decrease in estrogen levels during menopause. In conclusion, the fovea cones, through its sensitivity to estrogen and high energy consumption, may be very vulnerable to damage caused by a sudden changes in the concentration of estrogen in menopausal females. Such changes may result in cone degeneration, and thus a destroyed structure of the fovea, and may lead to the development of a hole in the fovea, as in the case of macular holes. This review revealed that under the decreasing influence of estrogen may cones play a key role with regard to the etiology of the development of macular holes. This aspect may be of strategic importance in prophylactic therapy for the prevention of the development of macular holes in premenopausal females or after ocular trauma.

Metabolic Engineering Design Strategies for Increasing Carbon Fluxes Relevant for Biosynthesis in Cyanobacteria.

Cyanobacteria are promising microbial cell factories for the direct production of biochemicals and biofuels from CO2. Through genetic and metabolic engineering, they can be modified to produce a variety of both natural and non-natural compounds. To enhance the yield of these products, various design strategies have been developed. In this chapter, strategies used to enhance metabolic fluxes towards common precursors used in biosynthesis, including pyruvate, acetyl-CoA, malonyl-CoA, TCA cycle intermediates, and aromatics, are discussed. Additionally, strategies related to cofactor availability and mixotrophic conditions for bioproduction are also summarize.

Increased Angiopoietin-1 and -2 levels in human vitreous are associated with proliferative diabetic retinopathy.

BACKGROUND: Diabetic retinopathy is a frequent complication of diabetes mellitus and a leading cause of blindness in adults. The objective of this study was to elucidate the diabetic retinopathy pathophysiology in more detail by comparing protein alterations in human vitreous of different diabetic retinopathy stages. METHODS: Vitreous samples were obtained from 116 patients undergoing pars plana vitrectomy. Quantitative immunoassays were performed of angiogenic factors (VEGF-A, PIGF, Angiopoietin-1, Angiopoietin-2, Galectin-1) as well as cytokines (IL-1ß, IL-8, IFN-γ, TNF-α, CCL3) in samples from control patients (patients who don't suffer from diabetes; n = 58) as well as diabetes mellitus patients without retinopathy (n = 25), non-proliferative diabetic retinopathy (n = 12), and proliferative diabetic retinopathy patients (n = 21). In addition, correlation analysis of protein levels in vitreous samples and fasting glucose values of these patients as well as correlation analyses of protein levels and VEGF-A were performed. RESULTS: We detected up-regulated levels of VEGF-A (p = 0.001), PIGF (p<0.001), Angiopoietin-1 (p = 0.005), Angiopoietin-2 (p<0.001), IL-1ß (p = 0.012), and IL-8 (p = 0.018) in proliferative diabetic retinopathy samples. Interestingly, we found a strong positive correlation between Angiopoietin-2 and VEGF-A levels as well as a positive correlation between Angiopoietin-1 and VEGF-A. CONCLUSION: This indicated that further angiogenic factors, besides VEGF, but also pro-inflammatory cytokines are involved in disease progression and development of proliferative diabetic retinopathy. In contrast, factors other than angiogenic factors seem to play a crucial role in non-proliferative diabetic retinopathy development. A detailed breakdown of the pathophysiology contributes to future detection and treatment of the disease.

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.

Changes of Subjective Symptoms and Tear Film Biomarkers following Femto-LASIK.

Femtosecond laser-assisted in situ keratomileusis (Femto-LASIK) represents a common treatment modality in refractive surgery and shows excellent results in terms of safety, efficacy, predictability, and long-term stability. However, patients may be affected by dry eye symptoms. The aim of this study was to identify a potential association between subjective dry eye symptoms, objective dry eye markers, and possible changes in the tear film, which could be a target for future therapy development. Therefore, clinical (dry eye) examinations (OSDI, Schirmer test, lissamine green and fluorescein staining, BUT, visual acuity) were carried out before LASIK as well as 5 and 90 days post-OP. The dry eye marker MMP-9, cytokines (IL-1ß, IL-8), and pain markers (NGF, CGRP) were quantified in tear samples with immunoassays. In addition, correlation analyses were performed. Clinical examinations revealed an upregulated OSDI score 5 days post-OP and an increased lissamine green staining score 90 days post-OP. Downregulated CGRP levels were noted 5 days post-OP, while other protein markers were not significantly altered after Femto-LASIK. Hence, Femto-LASIK surgery induced subjective symptoms like that of dry eye which could objectively rather be classified as Femto-LASIK-related discomfort. In the future, this could possibly be better detected and treated using pain markers such as CGRP.

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.

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.

Microglia Activation in Retinal Ischemia Triggers Cytokine and Toll-Like Receptor Response.

Mechanisms and progression of ischemic injuries in the retina are still incompletely clarified. Therefore, the time course of microglia activation as well as resulting cytokine expression and downstream signaling were investigated. Ischemia was induced in one eye by transiently elevated intraocular pressure (60 min) followed by reperfusion; the other eye served as a control. Eyes were processed for RT-qPCR and immunohistochemistry analyses at 2, 6, 12, and 24 h as well as at 3 and 7 days. Already 2 h after ischemia, more microglia/macrophages were in an active state in the ischemia group. This was accompanied by an upregulation of pro-inflammatory cytokines, like IL-1ß, IL-6, TNFα, and TGFß. Activation of TLR3, TLR2, and the adaptor molecule Myd88 was also observed after 2 h. NFκB revealed a wave-like activation pattern. In addition, an extrinsic caspase pathway activation was noted at early time points, while enhanced numbers of cleaved caspase 3+ cells could be observed in ischemic retinae throughout the study. Retinal ischemia induced an early and strong microglia/macrophage response as well as cytokine and apoptotic activation processes. Moreover, in early and late ischemic damaging processes, TLR expression and downstream signaling were involved, suggesting an involvement in neuronal death in ischemic retinae. Graphical Abstract.

Retina in a dish: Cell cultures, retinal explants and animal models for common diseases of the retina.

For many retinal diseases, including age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy (DR), the exact pathogenesis is still unclear. Moreover, the currently available therapeutic options are often unsatisfactory. Research designed to remedy this situation heavily relies on experimental animals. However, animal models often do not faithfully reproduce human disease and, currently, there is strong pressure from society to reduce animal research. Overall, this creates a need for improved disease models to understand pathologies and develop treatment options that, at the same time, require fewer or no experimental animals. Here, we review recent advances in the field of in vitro and ex vivo models for AMD, glaucoma, and DR. We highlight the difficulties associated with studies on complex diseases, in which both the initial trigger and the ensuing pathomechanisms are unclear, and then delineate which model systems are optimal for disease modelling. To this end, we present a variety of model systems, ranging from primary cell cultures, over organotypic cultures and whole eye cultures, to animal models. Specific advantages and disadvantages of such models are discussed, with a special focus on their relevance to putative in vivo disease mechanisms. In many cases, a replacement of in vivo research will mean that several different in vitro models are used in conjunction, for instance to analyze and validate causative molecular pathways. Finally, we argue that the analytical decomposition into appropriate cell and tissue model systems will allow making significant progress in our understanding of complex retinal diseases and may furthermore advance the treatment testing.

Organ Cultures for Retinal Diseases.

The successful development of novel therapies is closely linked with understanding the underlying pathomechanisms of a disease. To do so, model systems that reflect human diseases and allow for the evaluation of new therapeutic approaches are needed. Yet, preclinical animal studies often have limited success in predicting human physiology, pathology, and therapeutic responses. Moreover, animal testing is facing increasing ethical and bureaucratic hurdles, while human cell cultures are limited in their ability to represent in vivo situations due to the lack of the tissue microenvironment, which may alter cellular responses. To overcome these struggles, organ cultures, especially those of complex organs such as the retina, can be used to study physiological reactions to substances or stressors. Human and animal organ cultures are now well established and recognized. This mini-review discusses how retinal organ cultures can be used to preserve tissue architecture more realistically and therefore better represent disease-related changes. It also shows how molecular biological, biochemical, and histological techniques can be combined to investigate how anatomical localization may alter cellular responses. Examples for the use of retinal organ cultures, including models to study age-related macular degeneration (AMD), retinitis pigmentosa (RP), central artery occlusion (CRAO), and glaucoma are presented, and their advantages and disadvantages are discussed. We conclude that organ cultures significantly improve our understanding of complex retinal diseases and may advance treatment testing without the need for animal testing.

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.