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

Destructive effect of intravitreal heat shock protein 27 application on retinal ganglion cells and neurofilament

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

HSP27 induced glaucomatous damage in mice of young and advanced age

$\bf Introduction:$ 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 ($\it HSP27$). An intravitreal injection of $\it HSP27$ leads to glaucoma-like damage in rats. We now aimed to investigate if aged mice are more prone to this damage than younger ones. $\bf Methods:$ We intravitreally injected $\it HSP27$ 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. $\bf Results:$ 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 $\it HSP27$ mice at both ages. Comparing aged and young $\it HSP27$ mice, no differences in $\it Rbpms$ and $\it Pou4f1$ (RGCs) expression was detected, while the $\it Tubb3$ expression (neuronal cells) was significantly upregulated in aged $\it HSP27$ animals. Neither microglia/macrophages nor (resident) microglia counts revealed significant differences in $\it HSP27$ mice at both ages. Nevertheless, increased relative $\it Iba1$ and $\it Tmem119$ expression was detected in young and aged $\it HSP27$ mice. Aged $\it HSP27$ mice displayed a significantly lower $\it Iba1$ expression than young ones, whereas $\it Cd68$ levels were upregulated. A larger $\it GFAP+$ area and an upregulation of $\it GFAP$ expression in $\it HSP27$ animals of both ages indicated a macrogliosis. Also, elevated $\it Il1b$ and $\it Nos2$ expression levels were observed in young and aged $\it HSP27$ mice. However, only $\it Il1b$ levels were upregulated when comparing 7–8 months to 1–2 months old animals. A larger $\it HSP25+$ area was seen in aged $\it HSP27$ animals, while $\it Hspb2$ expression levels were downregulated in both $\it HSP27$ groups. The aged $\it HSP27$ group displayed an upregulated $\it Hspb2$ expression compared to young mice. Furthermore, a higher optic nerve degeneration score was noted in young and aged $\it HSP27$ groups. $\bf Discussion:$ These findings indicate that an intravitreal injection of $\it HSP27$ led to $\it RGC$ 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 $\it HSP27$ in the development of glaucoma

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

$\bf Background$ 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. $\bf Methods$ 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-1$\beta$, IL-8, IFN-$\gamma$, TNF-$\alpha$, 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. $\bf Results$ 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-1$\beta$ (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. $\bf Conclusion$ 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

Hypoxic processes induce complement activation via classical pathway in porcine neuroretinas

Considering the fact that many retinal diseases are yet to be cured, the pathomechanisms of these multifactorial diseases need to be investigated in more detail. Among others, oxidative stress and hypoxia are pathomechanisms that take place in retinal diseases, such as glaucoma, age-related macular degeneration, or diabetic retinopathy. In consideration of these diseases, it is also evidenced that the immune system, including the complement system and its activation, plays an important role. Suitable models to investigate neuroretinal diseases are organ cultures of porcine retina. Based on an established model, the role of the complement system was studied after the induction of oxidative stress or hypoxia. Both stressors led to a loss of retinal ganglion cells (RGCs) accompanied by apoptosis. Hypoxia activated the complement system as noted by higher $C3^{+}$ and $MAC^{+}$ cell numbers. In this model, activation of the complement cascade occurred via the classical pathway and the number of $C1q^{+}$ microglia was increased. In oxidative stressed retinas, the complement system had no consideration, but strong inflammation took place, with elevated $\it TNF$, $\it IL6$, and $\it IL8$ mRNA expression levels. Together, this study shows that hypoxia and oxidative stress induce different mechanisms in the porcine retina inducing either the immune response or an inflammation. Our findings support the thesis that the immune system is involved in the development of retinal diseases. Furthermore, this study is evidence that both approaches seem suitable models to investigate undergoing pathomechanisms of several neuroretinal diseases

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 ($H_{2}O_{2}$) in a porcine organ culture model. Retinal explants were cultivated for eight days, and $H_{2}O_{2}$ (500 $\mu$M, 3 h) induced the oxidative damage. CoQ10 therapy was applied (700 $\mu$M, 48 h). Retinal ganglion cells (RGCs) and microglia were examined immunohistologically in all groups (control, $H_{2}O_{2}$, $H_{2}O_{2}$ + CoQ10). Cellular, oxidative, and inflammatory genes were quantified via RT-qPCR. Strong RGC loss was observed with $H_{2}O_{2}$ ($\it p$ $\leq$ 0.001). CoQ10 elicited RGC protection compared to the damaged group at a histological ($\it p$ $\leq$ 0.001) and mRNA level. We detected more microglia cells with $H_{2}O_{2}$, but CoQ10 reduced this effect ($\it p$ = 0.004). Cellular protection genes ($\it NRF2$) against oxidative stress were stimulated by CoQ10 ($\it p$ $\leq$ 0.001). Furthermore, mitochondrial oxidative stress ($\it SOD2$) increased through $H_{2}O_{2}$ ($\it p$ = 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

Fewer functional deficits and reduced cell death after ranibizumab treatment in a retinal ischemia model

Retinal ischemia is an important factor in several eye disorders. To investigate the impact of VEGF inhibitors, as a therapeutic option, we studied these in a retinal ischemia animal model. Therefore, animals received bevacizumab or ranibizumab intravitreally one day after ischemia induction. Via electroretinography, a significant decrease in a- and b-wave amplitudes was detected fourteen days after ischemia, but they were reduced to a lesser extent in the ranibizumab group. Ischemic and bevacizumab retinae displayed fewer retinal ganglion cells (RGCs), while no significant cell loss was noted in the ranibizumab group. Apoptosis was reduced after therapy. More autophagocytotic cells were observed in ischemic and bevacizumab eyes, but not in ranibizumab eyes. Additionally, more microglia, as well as active ones, were revealed in all ischemic groups, but the increase was less prominent under ranibizumab treatment. Fewer cone bipolar cells were detected in ischemic eyes, in contrast to bevacizumab and ranibizumab-treated ones. Our results demonstrate a reduced apoptosis and autophagocytosis rate after ranibizumab treatment. Furthermore, a certain protection was seen regarding functionality, RGC, and bipolar cell availability, as well as microglia activation by ranibizumab treatment after ischemic damage. Thus, ranibizumab could be an option for treatment of retinal ischemic injury

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-1$\beta$, 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

Simultaneous complement response via lectin pathway in retina and optic nerve in an experimental autoimmune glaucoma model

Glaucoma is a multifactorial disease and especially mechanisms occurring independently from an elevated intraocular pressure (IOP) are still unknown. Likely, the immune system contributes to the glaucoma pathogenesis. Previously, IgG antibody depositions and retinal ganglion cell (RGC) loss were found in an IOP-independent autoimmune glaucoma model. Therefore, we investigated the possible participation of the complement system in this model. Here, rats were immunized with bovine optic nerve homogenate antigen (ONA), while controls (Co) received sodium chloride ($\it n$ = 5–6/group). After 14 days, RGC density was quantified on flatmounts. No changes in the number of RGCs could be observed at this point in time. Longitudinal optic nerve sections were stained against the myelin basic protein (MBP). We could note few signs of degeneration processes. In order to detect distinct complement components, retinas and optic nerves were labeled with complement markers at 3, 7, 14, and 28 days and analyzed. Significantly more C3 and MAC depositions were found in retinas and optic nerves of the ONA group. These were already present at day 7, before RGC loss and demyelination occurred. Additionally, an upregulation of C3 protein was noted via Western Blot at this time. After 14 days, quantitative real-time PCR revealed significantly more $\it C3$ mRNA in the ONA retinas. An upregulation of the lectin pathway-associated mannose-serine-protease-2 (MASP2) was observed in the retinas as well as in the optic nerves of the ONA group after 7 days. Significantly more MASP2 in retinas could also be observed via Western Blot analyses at this point in time. No effect was noted in regard to C1q. Therefore, we assume that the immunization led to an activation of the complement system via the lectin pathway in retinas and optic nerves at an early stage in this glaucoma model. This activation seems to be an early response, which then triggers degeneration. These findings can help to develop novel therapy strategies for glaucoma patients

Cytokine and complement response in the glaucomatous β\betaB1-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 $\beta$B1-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 $\beta$B1-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 C$_3$ and C$_5$ ($\it {Hc}$) were upregulated at 10 weeks. In accordance, more C$_3$+ and membrane attack complex$^{+}$ cells were observed in transgenic retinae. Further, the C5a receptor anaphylatoxin receptor ($\it {C5ar}$) and the complement component C5a receptor 1 ($\it {C5ar1}$; CD88) mRNA levels were upregulated in 10- and 15-week-old $\beta$B1-CTGF mice. Interestingly, all three activation routes of the complement system were elevated in βB1-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-$\gamma$ ($\it {Infg}$) were upregulated at 5 weeks, while $\it {Cxcl1}$ and $\it {Cxcl2}$ mRNA levels were upregulated at 10 and 15 weeks. The mRNA levels of the chemokines $\it {Cxcl10}$ were increased at all ages in $\beta$B1-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