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The aim of this research was to evaluate the effects of different lens types on the availability and efficacy of anti-inflammatory and antibiotic drugs. Three lens types were examined: (1) nonionic hydrogel lenses, (2) ionic hydrogel lenses, and (3) silicone hydrogel lenses. The lenses were incubated with (a) dexamethasone, (b) betamethasone, (c) bromophenacyl bromide, and (d) chloramphenicol. Drug availability was quantified by gradient HPLC, and chloramphenicol antibacterial activity was quantified by testing the inhibition of Salmonella typhimurium growth on agar. The lens allowing the most abundant passage of betamethasone was the ionic hydrogel lens, followed by the silicone hydrogel lens and nonionic hydrogel lens. The lens allowing the most abundant passage of dexamethasone was the ionic hydrogel lens, but only at 0.5 h and 1 h. Regarding chloramphenicol, the ionic hydrogel lens and silicone hydrogel lens allowed more abundant passage than the nonionic hydrogel lens. These results highlight the relevance of adapting lenses to anti-inflammatory therapy, thus allowing a personalized medical approach.

Glaucoma is a chronic, progressive and heterogeneous optic neuropathy which affects in the early stage the peripheral vision and then the central vision, leading to irreversible blindness. As known, in glaucoma the trabecular meshwork represent the main tissue which is impaired by chronic oxidative stress, aging and increase of intraocular pressure. Today, the lack of human-based models, with characteristics of high repeatability and reproducibility as well, called for an high-quality in vitro model with a good degree of resemblance for the tissue or organ of interest as a basis for new drug testing. Our team has been committed to this purpose by assessment of an in vitro 3D TM human-based model, closer to in vivo, using millifluidic technology, to better identify the key events underlying the pathogenesis of glaucoma and to evaluate new therapies targeted at disease treatment and prevention.

Aloe vera extracts have been used for drug therapy due to their antimicrobial, anti-inflammatory, and epithelia-regenerative actions. Considering the possible adverse side effects on the corneal surface of anti-microbial agent administration after ocular surgery, it is of interest to identify the ability of Aloe vera extract release from a therapeutic lens to prevent ocular irritation. The aim of this research was to evaluate the effects of different lens types on the availability and efficacy of releasing Aloe vera extracts in which they are soaked. Two lens types were examined: (1) non-ionic hydrogel lenses and (2) ionic hydrogel lenses. Aloe vera availability was quantified by LC&ndash, MS, and activity was quantified by a spectrophotometer (Nanodrop) at different times: (a) 30 min, (b) 60 min, (c) 2 h, (d) 6 h, and (e) 24 h. The lens allowing for the most abundant passage of Aloe vera extracts was the ionic hydrogel lens, followed by the non-ionic hydrogel lens. The obtained results demonstrate that the best lens allowing for the high and stable release of Aloe vera extract to the corneal surface are those composed of ionic hydrogels.

Neurodegeneration can be defined as progressive cell damage to nervous system cells, and more specifically to neurons, which involves morphologic alterations and progressive loss of function until cell death. Glaucoma exhibits many aspects of neurodegenerative disease. This review examines the pathogenesis of glaucoma, comparing it with that of Alzheimer's disease (AD) and Parkinson's disease (PD), highlighting their common features. Indeed, in all three diseases there are not only the same types of pathogenic events, but also similarities of temporal cadences that determine neuronal damage. All three age-related illnesses have oxidative damage and mitochondrial dysfunction as the first pathogenic steps. The consequence of these alterations is the death of visual neurons in glaucoma, cognitive neurons in AD and regulatory motor neurons (substantia nigra) in PD. The study of these common pathogenic events (oxidative stress, mitochondrial dysfunction, protein degradation, apoptosis and autophagy) leads us to consider common therapeutic strategies for the treatment and prevention of these diseases. Also, examination of the genetic aspects of the pathways involved in neurodegenerative processes plays a key role in shedding light on the details of pathogenesis and can suggest new treatments. This review discusses the common molecular aspects involved in these three oxidative-stress and age-related diseases.

Glaucoma is a chronic neurodegenerative disease characterized by retinal ganglion cell loss. Although significant advances in ophthalmologic knowledge and practice have been made, some glaucoma mechanisms are not yet understood, therefore, up to now there is no effective treatment able to ensure healing. Indeed, either pharmacological or surgical approaches to this disease aim in lowering intraocular pressure, which is considered the only modifiable risk factor. However, it is well known that several factors and metabolites are equally (if not more) involved in glaucoma. Oxidative stress, for instance, plays a pivotal role in both glaucoma onset and progression because it is responsible for the trabecular meshwork cell damage and, consequently, for intraocular pressure increase as well as for glaucomatous damage cascade. This review at first shows accurately the molecular-derived dysfunctions in antioxidant system and in mitochondria homeostasis which due to both oxidative stress and aging, lead to a chronic inflammation state, the trabecular meshwork damage as well as the glaucoma neurodegeneration. Therefore, the main molecular events triggered by oxidative stress up to the proapoptotic signals that promote the ganglion cell death have been highlighted. The second part of this review, instead, describes some of neuroprotective agents such as polyphenols or polyunsaturated fatty acids as possible therapeutic source against the propagation of glaucomatous damage.

Glaucoma is the second leading cause of blindness worldwide. Currently, glaucoma treatments aim to lower intraocular pressure by decreasing aqueous humor production or increasing aqueous humor outflow through pharmacological approaches or trabeculectomy. The lack of an effective cure requires new therapeutic strategies. We compared the bio­logical responses of a three-dimensional trabecular meshwork model with or without perfusion bioreactor technology to better understand the early molecular changes induced by prolonged oxidative stress conditions induced by repeated daily peroxide exposure. We used standard 3D cultures of trabecular meshwork cells in Matrigel cultured under either static and dynamic conditions for one week. We studied changes in F-actin expression and organization in the cells, cellular metabolic activity, proinflammatory gene expression, expression of pro- and anti-apoptotic proteins, PARP-1 cleavage, and NFκB activation in the model. We demonstrate that the dynamic conditions improve the adaptive behavior of 3D trabecular meshwork cultures to chronic oxidative stress via offsetting pathway activation.

Many diseases are related to age, among these neurodegeneration is particularly important. Alzheimer's disease Parkinson's and Glaucoma have many common pathogenic events including oxidative damage, Mitochondrial dysfunction, endothelial alterations and changes in the visual field. These are well known in the case of glaucoma, less in the case of neurodegeneration of the brain. Many other molecular aspects are common, such as the role of endoplasmic reticulum autophagy and neuronal apoptosis while others have been neglected due to lack of space such as inflammatory cytokine or miRNA. Moreover, the loss of specific neuronal populations, the induction of similar mechanisms of cell injury and the deposition of protein aggregates in specific anatomical areas are very similar events between these diseases. Intracellular and/or extracellular accumulation of protein aggregates is a key feature of many neurodegenerative disorders. The existence of abnormal protein aggregates has been documented in the RGCs of glaucomatous patients such as the anomalous Tau protein or the β-amyloid accumulations. Intra-cell catabolic processes also appear to be common in both glaucoma and neurodegeneration. They also help us to understand how the basis between these diseases is common and how the visual aspects can be a serious problem for those who are affected.

Many diseases are related to age, among these neurodegeneration is particularly important. Alzheimer's disease Parkinson's and Glaucoma have many common pathogenic events including oxidative damage, Mitochondrial dysfunction, endothelial alterations and changes in the visual field. These are well known in the case of glaucoma, less in the case of neurodegeneration of the brain. Many other molecular aspects are common, such as the role of endoplasmic reticulum autophagy and neuronal apoptosis while others have been neglected due to lack of space such as inflammatory cytokine or miRNA. Moreover, the loss of specific neuronal populations, the induction of similar mechanisms of cell injury and the deposition of protein aggregates in specific anatomical areas are very similar events between these diseases. Intracellular and/or extracellular accumulation of protein aggregates is a key feature of many neurodegenerative disorders. The existence of abnormal protein aggregates has been documented in the RGCs of glaucomatous patients such as the anomalous Tau protein or the β-amyloid accumulations. Intra-cell catabolic processes also appear to be common in both glaucoma and neurodegeneration. They also help us to understand how the basis between these diseases is common and how the visual aspects can be a serious problem for those who are affected.

A physiologically relevant in vitro human-based model could be the ‘gold standard’ to clarify the pathological steps involved in glaucoma onset. In this regard, human 3D cultures may represent an excellent starting point to achieve this goal. Indeed, the 3D matrix allows to re-create the in vivo-like tissue architecture, maintaining its functionality and cellular behaviour, compared to the 2D model. Thus, we propose a comparison between the 2D and 3D in vitro models of human trabecular meshwork cells in terms of cellular responses after chronic stress exposure. Our results showed that 3D-cells are more sensitive to intracellular reactive oxidative specie production induced by hydrogen peroxide treatment, compared to 2D cultures. Additionally, in 3D cultures a more accurate regulation of the apoptosis trigger and cell adaptation mechanisms was detected than in 2D models. In line with these findings, the 3D-HTMC model shows the ability to better mimic the in vivo cell behaviour in adaptive responses to chronic oxidative stress than 2D.

Polyphenols, with anti-oxidant properties, counteract oxidative stress effects. Increasing evidence has found oxidative stressto be the main risk factor for trabecular meshwork (TM) damage, leading to high-tension glaucoma. Topical anti-oxidants could represent a new target for glaucoma treatment. Our aim is to investigate the protective mechanisms on a human TM culture of a patented polyphenol and fatty acid (iTRAB&reg, )formulation in response to oxidative stress using an advanced invitromodel consisting of 3D-human TM cells, embedded in a natural hydrogel, and a milli-scaled multi-organ device model for constantdynamic conditions. The 3D-human TM cells(3D-HTMCs) were treated daily with 500 &micro, M H2O2or 500 &micro, M H2O2and 0.15% iTRAB(m/v) for 72 h, and molecular differences in the intracellular reactive oxygen species (iROS), state of the cells, activation of the apoptosis pathway and NF-kB and the expression ofinflammatory and fibrotic markers wereanalyzed at different time-points.Concomitant exposure significantly reduced iROS and restored TM viability, iTRAB&reg, having a significant inhibitory effect on the apoptotic pathway, activation of NF-&kappa, B, induction of pro-inflammatory (IL-1&alpha, IL-1&szlig, and TNF&alpha, ) and pro-fibrotic (TGF&beta, ) cytokines and the matrix metalloproteinase expressions. It is clear that this specific anti-oxidant provides a valid TM protection, suggesting iTRAB&reg, could be an adjuvant therapy in primary open-angle glaucoma (POAG).

Glaucoma is a degenerative disease of the eye. Both the anterior and posterior segments of the eye are affected, extensive damage being detectable in the trabecular meshwork and the inner retina-central visual pathway complex. Oxidative stress is claimed to be mainly responsible for molecular damage in the anterior chamber. Indeed, oxidation harms the trabecular meshwork, leading eventually to endothelial cell decay, tissue malfunction, subclinical inflammation, changes in the extracellular matrix and cytoskeleton, altered motility, reduced outflow facility and (ultimately) increased IOP. Moreover, free radicals are involved in aging and can be produced in the brain (as well as in the eye) as a result of ischemia, leading to oxidation of the surrounding neurons. Glaucoma-related cell death occurs by means of apoptosis, and apoptosis is triggered by oxidative stress via (a) mitochondrial damage, (b) inflammation, (c) endothelial dysregulation and dysfunction, and (d) hypoxia. The proteomics of the aqueous humor is significantly altered in glaucoma as a result of oxidation-induced trabecular damage. Those proteins whose aqueous humor levels are increased in glaucoma are biomarkers of trabecular meshwork impairment. Their diffusion from the anterior to the posterior segment of the eye may be relevant in the cascade of events triggering apoptosis in the inner retinal layers, including the ganglion cells.

The trabecular meshwork (TM) plays an important role in high-tension glaucomas. Indeed, the TM is a true organ, through which the aqueous humor flows from the anterior chamber to Schlemm's canal (SC). Until recently, the TM, which is constituted by endothelial-like cells, was described as a kind of passive filter. In reality, it is much more. The cells delineating the structures of the collagen framework of the TM are endowed with a cytoskeleton, and are thus able to change their shape. These cells also have the ability to secrete the extracellular matrix, which expresses proteins and cytokines, and are capable of phagocytosis and autophagy. The cytoskeleton is attached to the nuclear membrane and can, in millionths of a second, send signals to the nucleus in order to alter the expression of genes in an attempt to adapt to biomechanical insult. Oxidative stress, as happens in aging, has a deleterious effect on the TM, leading eventually to cell decay, tissue malfunction, subclinical inflammation, changes in the extracellular matrix and cytoskeleton, altered motility, reduced outflow facility, and (ultimately) increased IOP. TM failure is the most relevant factor in the cascade of events triggering apoptosis in the inner retinal layers, including ganglion cells. J. Cell. Physiol. 231: 1876-1893, 2016. © 2016 Wiley Periodicals, Inc.

Introduction Primary open angle glaucoma (POAG) is a progressive optic neuropathy characterized by impaired aqueous outflow and extensive remodeling in the trabecular meshwork (TM). The aim of this study was to characterize and compare the expression patterns of selected proteins belonging to the tissue remodeling, inflammation and growth factor pathways in ex vivo glaucomatous and post-mortem TMs using protein-array analysis. Methods TM specimens were collected from 63 white subjects, including 40 patients with glaucoma and 23 controls. Forty POAG TMs were collected at the time of surgery and 23 post-mortem specimens were from non-glaucomatous donor sclerocorneal tissues. Protein profiles were evaluated using a chip-based array consisting of 60 literature-selected antibodies. Results A different expression of some factors was observed in POAG TMs with respect to post-mortem specimens, either in abundance (interleukin [IL]10, IL6, IL5, IL7, IL12, IL3, macrophage inflammatory protein [MIP]1δ/α, vascular endothelial growth factor [VEGF], transforming growth factor beta 1 [TGFβ1], soluble tumor necrosis factor receptor I [sTNFRI]) or in scarcity (IL16, IL18, intercellular adhesion molecule 3 [ICAM3], matrix metalloproteinase-7 [MMP7], tissue inhibitor of metalloproteinase 1 [TIMP1]). MMP2, MMP7, TGFβ1, and VEGF expressions were confirmed by Western blot, zymography, and polymerase chain reaction. No difference in protein profile expression was detected between glaucomatous subtypes. Conclusion The analysis of this small TM population highlighted some proteins linked to POAG, some previously reported and others of new detection (IL7, MIPs, sTNFαRI). A larger POAG population is required to select promising disease-associated biomarker candidates. Funding This study was partially supported by the Fondazione Roma, the Italian Ministry of Health and the “National 5xMille 2010 tax donation to IRCCS-G.B. Bietti Foundation”. Electronic supplementary material The online version of this article (doi:10.1007/s12325-016-0285-x) contains supplementary material, which is available to authorized users.

Primary open-angle glaucoma (POAG) is the second leading cause of irreversible blindness worldwide. Increasing evidence suggests oxidative damage and immune response defects are key factors contributing to glaucoma onset. Indeed, both the failure of the trabecular meshwork tissue in the conventional outflow pathway and the neuroinflammation process, which drives the neurodegeneration, seem to be linked to the age-related over-production of free radicals (i.e., mitochondrial dysfunction) and to oxidative stress-linked immunostimulatory signaling. Several previous studies have described a wide range of oxidative stress-related makers which are found in glaucomatous patients, including low levels of antioxidant defences, dysfunction/activation of glial cells, the activation of the NF-κB pathway and the up-regulation of pro-inflammatory cytokines, and so on. However, the intraocular pressure is still currently the only risk factor modifiable by medication or glaucoma surgery. This present review aims to summarize the multiple cellular processes, which promote different risk factors in glaucoma including aging, oxidative stress, trabecular meshwork defects, glial activation response, neurodegenerative insults, and the altered regulation of immune response.

In this manuscript, which appeared in ALTEX 37, 265-274 (doi: 10.14573/altex.1909262), the affiliation of Stefania Vernazza should read: Stefania Vernazza 5# 5 IRCCS-Fondazione Bietti, Rome, Italy and the address for correspondence should read: Stefania Vernazza, PhD, IRCCS, Fondazione Bietti via Livenza 3, 00198 Rome, Italy (stefania.vernazza@yahoo.it).

PurposeOxidative damage to the trabecular meshwork (TM) represents one of the pathogenic mechanisms leading to primary open angle glaucoma (POAG). Glutathione S-transferase mu 1 (GSTM1) may neutralizes reactive oxygen species protecting the TM. The present paper investigates the prevalence of GSTM1 null genotype in an Italian population, and its association with POAG treated either medically or surgically.MethodsIn a case-control study, the GSTM1 genotype was identified in POAGs and controls. The POAGs patients were divided in two groups: medical POAGs and surgical POAGS. Medical POAGs consisted of patients with a well-controlled intraocular pressure (IOP) by IOP-lowering medications and a stable visual field (VF). Patients with an uncontrolled IOP and a progressing VF that were submitted to incisional surgery formed the surgical POAGs’ group.ResultsWe enrolled 104 medical POAGs, 158 surgical POAGs and 263 Controls. No significative differences between the groups existed regarding age and gender (p=0.275 and p=0.950, respectively). All the enrolled subjects were Caucasian of Italian descents. The GSTM1 null genotype was identified in 57 (45.2%) medical POAGs, 91 (57.6%) surgical POAGs and, 119 (45.3%) controls (p=0.033). The association between medical POAG and GSTM1 null status was non-significant (OR= 1.44, 95% IC = 0.86 to 2.39) whereas the association was significant for surgical POAGs (OR= 2.01, 95% IC= 1.26 to 3.21)ConclusionsOur results showed an association between the GSTM1 null genotype and glaucoma that require surgery in an Italian population. GSTM1 null genotype detection may help to identify high-risk glaucoma patients that require a closer follow-up and a more aggressive treatments.

The human eye is constantly exposed to sunlight and artificial lighting. Exogenous sources of reactive oxygen species (ROS) such as UV light, visible light, ionizing radiation, chemotherapeutics, and environmental toxins contribute to oxidative damage in ocular tissues. Long-term exposure to these insults places the aging eye at considerable risk for pathological consequences of oxidative stress. Furthermore, in eye tissues, mitochondria are an important endogenous source of ROS. Over time, all ocular structures, from the tear film to the retina, undergo oxidative stress, and therefore, the antioxidant defenses of each tissue assume the role of a safeguard against degenerative ocular pathologies. The ocular surface and cornea protect the other ocular tissues and are significantly exposed to oxidative stress of environmental origin. Overwhelming of antioxidant defenses in these tissues clinically manifests as pathologies including pterygium, corneal dystrophies, and endothelial Fuch's dystrophy. The crystalline lens is highly susceptible to oxidative damage in aging because its cells and their intracellular proteins are not turned over or replaced, thus providing the basis for cataractogenesis. The trabecular meshwork, which is the anterior chamber tissue devoted to aqueous humor drainage, has a particular susceptibility to mitochondrial oxidative injury that affects its endothelium and leads to an intraocular pressure increase that marks the beginning of glaucoma. Photo-oxidative stress can cause acute or chronic retinal damage. The pathogenesis of age-related macular degeneration involves oxidative stress and death of the retinal pigment epithelium followed by death of the overlying photoreceptors. Accordingly, converging evidence indicates that mutagenic mechanisms of environmental and endogenous sources play a fundamental pathogenic role in degenerative eye diseases.

Proteomic analyses as applied to ocular aqueous humor provide evidence that this approach may be used to identify ocular disease with particular reference to cataract and glaucoma. Protein alterations bear pathogenic relevance for disease development. Among the different methods available, antibody microarray seems to be the most readily transferable to the clinic. However, this method still bears some limitations, such as the relatively small number of proteins analyzed and the poor specificity. Proteomic analysis is able to depict the pathogenesis of common ocular diseases and to produce data of both diagnostic and prognostic relevance. Proteome alterations detected in the aqueous humor of glaucomatous patients reflect degeneration occurring in target tissues - that is, the trabecular meshwork, retina and optic nerve head. Performed studies indicate good performances of aqueous humor analysis by antibody microarray for glaucoma diagnosis. Future development is addressed to improve antibody microarray specificity and to set up minimally invasive procedures for aqueous humor sampling.

Oxidative damage plays a pathogenic role in various chronic degenerative diseases. Oxidative damage targeting trabecular meshwork (TM) cells as a consequence of mitochondrial damage is a pathogenic mechanism for glaucoma, the most common cause of irreversible blindness worldwide. Consequences of oxidative damage are attenuated by endocellular activities involved in scavenging reactive oxidative species and DNA repair. Selected bacterial genes are highly efficient at protecting cells from oxidative DNA damage. This situation occurs for Escherichia coli formamidopyrimidine DNA glycosylase (FPG), a major DNA glycosylase that repairs oxidatively damaged DNA. Accordingly, this study was aimed at transfecting human TM cells (HTMC) with Fpg in order to increase their resistance to oxidative damage. This study demonstrates that it is feasible to increase resistance of HTMC to endogenous oxidative damage by gene transfection. These findings bear relevance for primary and secondary prevention of degenerative glaucomas and other degenerative diseases where oxidative damage plays a pathogenic role.

Polyunsaturated fatty acids (PUFA) are known to have numerous beneficial effects, owing to their anti-inflammatory and antioxidant properties. From a metabolic standpoint, the mitochondria play a fundamental role in cellular homeostasis, and oxidative stress can affect their functioning. Indeed, the mitochondria are the main source of ROS, and an imbalance between ROS and antioxidant defenses leads to oxidative stress. In addition, aging, the decline of cellular functions, and continual exposure to light underlie many diseases, particularly those of the eye. Long-term exposure to insults, such as UV light, visible light, ionizing radiation, chemotherapeutics, and environmental toxins, contribute to oxidative damage in ocular tissues and expose the aging eye to considerable risk of pathological consequences of oxidative stress. Ample antioxidant defenses responsible for scavenging free radicals are essential for redox homeostasis in the eye, indeed, eye tissues, starting from the tear film, which normally are exposed to high oxygen levels, have strong antioxidant defenses that are efficient for protecting against ROS-related injuries. On the contrary, instead, the trabecular meshwork is not directly exposed to light and its endothelial cells are poorly equipped with antioxidant defenses. All this makes the eye a target organ of oxidative damage. This review focuses on the role of the polyunsaturated fatty acids in the human eye, particularly in such pathologies as dry eye, glaucoma, and macular degeneration, in which dietary PUFA supplementation can be a valid therapeutic aid.

Glaucoma targets a variety of different tissues located in both anterior (e.g., trabecular meshwork) and posterior (e.g., optic nerve head) ocular segments. The transmission of damage between these structures cannot be simply ascribed to intraocular pressure increase. Recent experimental findings provide evidence for the involvement of molecular mediators including proteins and microRNAs. Aqueous humor protein composition is characteristically altered during glaucoma progression. Immunohistochemistry analyses indicate that proteins characterizing glaucomatous aqueous humor are released by damaged trabecular meshwork. This feature incudes (a) Nestin, involved in stem cell recruitment and glial cell activation; (b) A Kinase anchor protein, released as consequence of mitochondrial damage and Rho activation establishing cell shape and motility; (c) Actin related protein 2/3 complex, involved in actin polymerization and cell shape maintenance. As established both in vitro and in glaucomatous aqueous humor, trabecular meshwork cells damaged by oxidative stress release extracellular microRNAs inducing glial cell activation, an established pathogenic mechanism in neurodegenerative diseases. Released microRNAs include miR-21 (apoptosis), miR-450 (cell aging, maintenance of contractile tone), miR-107 (Nestin expression, apoptosis), miR-149 (endothelia and extracellular matrix homeostasis). Experimental evidences indicate that the uveoscleral pathway, via suprachoroidal space, can provide a potential route of access from the anterior region to the posterior segment of the eye and could represent the path followed by biologic mediators to reach the inner layer of the peripapillary retina and transmit damage signals from the anterior to posterior segment during glaucoma course.

Primary open angle glaucoma is a multi-tissue disease that targets, in an ascending order, the trabecular meshwork, the optic nerve head, the lateral geniculate nuclei, and the visual cortex. Oxidative stress and vascular damage play major roles in triggering apoptotic cell loss in these tissues. Molecular alterations occurring in the ocular anterior chamber during the early course of glaucoma trigger this cell loss. These molecular events are mainly of endogenous origin and related to the long-term accumulation of oxidative damages arising from mitochondrial failure and endothelial dysfunction. This situation results in decreased antioxidant defences in aqueous humour and apoptosis activation in trabecular meshwork cells as triggered by severe mitochondrial damage altering tissue function and integrity. The presence of neural proteins in glaucomatous aqueous humour indicate that a molecular interconnection exists between the anterior and the posterior chamber tissues. Trabecular meshwork and lamina cribrosa share a common neuro-ectodermal embryological, which contribute to explain the interconnection between anterior and the posterior chamber during glaucoma pathogenesis. During glaucoma, proteins deriving from the damage occurring in endothelial trabecular meshwork cells are released into aqueous humour. Accordingly, aqueous humour composition is characterised in glaucomatous patients by the presence of proteins deriving from apoptosis activation, mitochondrial damage, loss of intercellular connections, antioxidant decrease. Many questions remain unanswered, but molecular events illuminate TM damage and indicate that trabecular cell protection plays a role in the treatment and prevention of glaucoma.

Oxidative stress plays an important role in glaucoma. Some preservatives of anti-glaucoma drugs, commonly used in glaucoma therapy, can prevent or induce oxidative stress in the trabecular meshwork. The aim of this study is to evaluate cellular and molecular damage induced in trabecular meshwork by preservatives contained in anti-glaucoma drugs. Cell viability (MTT test), DNA fragmentation (Comet test), oxidative DNA damage (8-oxo-dG), and gene expression (cDNA microarray) have been evaluated in trabecular meshwork specimens and in human trabecular meshwork cells treated with benzalkonium chloride, polyQuad, purite, and sofzia-like mixture. Moreover, antimicrobial effectiveness and safety of preservative contents in drugs was tested. In ex vivo experiments, benzalkonium chloride and polyQuad induced high level of DNA damage in trabecular meshwork specimens, while the effect of purite and sofzia were more attenuated. The level of DNA fragmentation induced by benzalkonium chloride was 2.4-fold higher in subjects older than 50 years than in younger subjects. Benzalkonium chloride, and polyQuad significantly increased oxidative DNA damage as compared to sham-treated specimens. Gene expression was altered by benzalkonium chloride, polyQuad, and purite but not by sofzia. In in vitro experiments, benzalkonium chloride and polyQuad dramatically decreased trabecular meshwork cell viability, increased DNA fragmentation, and altered gene expression. A lesser effect was also exerted by purite and sofzia. Genes targeted by these alterations included Fas and effector caspase-3. The efficacy of the preservatives in inhibiting bacterial growth increased the adverse effects in trabecular meshwork in terms of DNA damage and alteration of gene expression. Presented data indicates the delicate balance between efficacy and safety of drug preservatives as not yet optimized.

DNA damage is related to a variety of degenerative diseases such as cancer, atherosclerosis and neurodegenerative diseases, depending on the tissue affected. Increasing evidence indicates that reactive oxygen species (ROS) play a key role in the pathogenesis of primary open angle glaucoma (POAG), the main cause of irreversible blindness worldwide. Oxidative DNA damage is significantly increased in the ocular epithelium regulating aqueous humor outflow, i.e., the trabecular meshwork (TM), of glaucomatous patients compared to controls. The pathogenic role of ROS in glaucoma is supported by various experimental findings, including (a) resistance to aqueous humor outflow is increased by hydrogen peroxide by inducing TM degeneration; (b) TM possesses remarkable antioxidant activities, mainly related to superoxide dismutase-catalase and glutathione pathways that are altered in glaucoma patients; and (c) intraocular-pressure increase and severity of visual-field defects in glaucoma patients parallel the amount of oxidative DNA damage affecting TM. Vascular alterations, which are often associated with glaucoma, could contribute to the generation of oxidative damage. Oxidative stress, occurring not only in TM but also in retinal cells, appears to be involved in the neuronal cell death affecting the optic nerve in POAG. The highlighting of the pathogenic role of ROS in POAG has implications for the prevention of this disease as indicated by the growing number of studies using genetic analyses to identify susceptible individuals and of clinical trials testing the efficacy of antioxidant drugs for POAG management.

The connection between Helicobacter pylori (Hp) infection and eye diseases has been increasingly reported in the literature and in active research. The implication of this bacterium in chronic eye diseases, such as blepharitis, glaucoma, central serous chorioretinopathy and others, has been hypothesized. Although the mechanisms by which this association occurs are currently unknown, this review describes shared pathogenetic mechanisms in an attempt to identify a lowest common denominator between eye diseases and Hp infection. The aim of this review is to assess whether different studies could be compared and to establish whether or not Hp infection and Eye diseases share common pathogenetic aspects. In particular, it has been focused on oxidative damage as a possible link between these pathologies. Text word search in Medline from 1998 to July 2014. 152 studies were included in our review. Were taken into considerations only studies that related eye diseases more frequent and/or known. Likely oxidative stress plays a key role. All of the diseases studied seem to follow a common pattern that implicates a cellular response correlated with a sublethal dose of oxidative stress. These alterations seem to be shared by both Hp infections and ocular diseases and include the following: decline in mitochondrial function, increases in the rate of reactive oxygen species production, accumulation of mitochondrial DNA mutations, increases in the levels of oxidative damage to DNA, proteins and lipids, and decreases in the capacity to degrade oxidatively damaged proteins and other macromolecules. This cascade of events appears to repeat itself in different diseases, regardless of the identity of the affected tissue. The trabecular meshwork, conjunctiva, and retina can each show how oxidative stress may acts as a common disease effector as the Helicobacter infection spreads, supported by the increased oxidative damage and other inflammation.

Glaucoma is a disease known since the time of Hippocrates. Today, the term glaucoma refers to a spectrum of neurodegenerative diseases that have in common progressive optical atrophy resulting from the apoptosis of retinal ganglion cells, and axonal atrophy and degeneration extending to the visual areas of the brain cortex, finally leading to the characteristic optical-cup neuropathy and irreversible visual loss. The action of many factors, including aging, genetic predisposition, exogenous environmental and endogenous factors, is necessary for the development of glaucoma. In addition to ganglion cell loss, most glaucoma types are characterized by high intraocular pressure. This is due to the damage that occurs in the trabecular meshwork, a key region in the pathogenesis of high-pressure glaucoma. In normal-pressure glaucoma, the pathogenesis is different, with vascular factors playing a very important role.

Aim—To evaluate daily tonometric curves after cataract surgery in patients with cataract only and in patients with cataract and glaucoma. Methods—108 patients scheduled for cataract surgery were randomly allocated to two groups: 57 patients with cataract only (normal) and 51 with cataract and primary open angle glaucoma (POAG). All patients underwent extracapsular cataract extraction (ECCE) (manual technique with long wound), phacoemulsification (automated technique with short wound), or nucleus capture (manual technique with short wound). Intraocular pressure (IOP) was measured by Goldmann tonometry in all patients every 2 hours for 12 hours before the operation and at 1 and 6 months postoperatively. Results—79 patients completed the 6 month examination. ECCE resulted in greater reductions in IOP than the other procedures (ECCE: 27% and 36% in normal patients and those with POAG, respectively; nucleus capture: 20% and 31%, respectively; phacoemulsification: 19% and 22%, respectively). The fluctuations in IOP before and after surgery were not statistically significant. Conclusion—Cataract surgery in normal patients reduces IOP but does not eliminate fluctuations which are directly proportional to the IOP value and result partly from circadian rhythms. This important finding might influence our approach to treatment of patients with glaucoma. (Br J Ophthalmol 2001;85:24‐29)

Purpose: The authors wished to verify the analgesic action of 0.1% indomethacin in a water-based solution on patients affected by traumatic corneal abrasions. Methods: 347 patients affected by traumatic corneal abrasions, having been randomly divided into 2 groups on the basis of the administration of indomethacin, were evaluated at 30 min, 12 h and 24 h after the initial treatment of the abrasion. The level of pain experienced was evaluated on a verbal pain scale and the healing time was evaluated relative to the dimension of the abrasion. Results: The pain level was initially overwhelming for both groups: p = 0.737; at successive check-ups it was possible to verify a reduction of the symptomatology, with a more pronounced decrease in pain in the group treated with indomethacin (p < 0.0001), which also demonstrated a lower sensitivity to pain in the case of larger lesions (p < 0.0001). There was no difference in the healing time between groups, and the reduction of pain is not correlated with corneal anesthesia and healing time. Conclusions: Our study highlighted the efficacy of indomethacin as a pain reducer for acute corneal pathology and suggested that the medication may act on the corneal nociceptors in a qualitative way.

Primary open-angle glaucoma is a multifactorial disease that affects the retinal ganglion cells, but currently its therapy is to lower the eye pressure. This indicates a definite involvement of the trabecular meshwork, key region in the pathogenesis of glaucoma. This is the first target of glaucoma, and its functional complexity is a real challenge to search. Its functions are those to allow the outflow of aqueous humor and not the reflux. This article describes the morphological and functional changes that happen in anterior chamber. The “primus movens” is oxidative stress that affects trabecular meshwork, particularly its endothelial cells. In these develops a real mitochondriopaty. This leads to functional impotence, the trabecular meshwork altering both motility and cytoarchitecture. Its cells die by apoptosis, losing barrier functions and altering the aqueous humor outflow. All the morphological alterations occur that can be observed under a microscope. Intraocular pressure rises and the malfunctioning trabecular meshwork endotelial cells express proteins that completely alter the aqueous humor. This is a liquid whose functional proteomics complies with the conditions of the trabecular meshwork. Indeed, in glaucoma, it is possible detect the presence of proteins which testify to what occurs in the anterior chamber. There are six classes of proteins which confirm the vascular endothelium nature of the anterior chamber and are the result of the morphofunctional trabecular meshwork decay. It is possible that, all or in part, these proteins can be used as a signal to the posterior pole.

The purpose of this work was to investigate the expression of glutamine synthase (GS), nitric oxide synthase (NOS) superoxide dismutase (SOD) and glutathione transferase (GST) in the aqueous humor of patients with primary open angle glaucoma and controls. Aqueous humor proteome was analyzed by antibody microarray. The expression of tested proteins was detected by protein Cy3/Cy5 labeling, column purification and hybridization on antibody-spotted glass microarray. Fluorescent signals were detected by fluorescence laser scanning. Aqueous humor levels of SOD as well as of GST were significantly lower (2.0- and 2.2-fold, p < 0.01) among patients than controls; both NOS and GS expression were significantly higher (2.2- and 2.6 fold, p < 0.01) among patients than controls. Our data showed substantial differences of GS, NOS2, SOD and GST aqueous humor levels between glaucomatous patients and controls as measured by antibody microarray technology. The overproduction of NO through inducible NOS can form toxic products and change the metabolic conditions of the TM. The GS over-expression might be related to neuronal injury or to the potential role of glutamate as a modulator in the ciliary body signaling. The reduced expression of the antioxidant enzymes SOD and GST could aggravate the unbalance between both oxygen- and nitrogen-derived free radicals production and detoxification. Based on our results, GS, NOS2, SOD and GST as measured by antibody microarray technology may be useful oxidative markers in aqueous humor of glaucomatous patients.

The glaucomas is a group of complex and heterogeneous ocular diseases representing the second leading cause of blindness, and almost 75 million people are affected worldwide (Quigley 1999) being a major issue for public health. The prevalence of glaucoma increases with age (Friedman et al. 2006). The reported prevalence among whites in their 80s varies widely across these studies, with estimates as low as 1.9%3 to as high as 8.8%. Glaucoma is a syndrome characterized by a progressive optical atrophy resulting from the apoptosis of the retinal ganglion cells (RGCs). Growing evidence obtained from clinical and experimental studies over the past decade strongly suggests the involvement of the reactive oxygen species (ROS) in glaucoma. Free radicals can directly induce neuronal death by a protease and phosphatase-gated mechanism distinct from apoptosis (See and Loeffler 2001). In glaucoma free radicals may damage the trabecular meshwork (TM) (Sacca et al. 2005) while in the posterior segment of the eye the process of apoptotic retinal ganglion cell death starts with exposure of glial cells to elevated concentrations of free radicals (Nakazawa et al. 2006). The final neurological damage results in progressive RGCs death, axon atrophy and degeneration also extending to the brain cortex (visual areas) finally leading to the characteristic optical-cup neuropathy and irreversible visual loss (Weber and Harman 2005) (Yucel et al. 2000). In addition to the loss of the ganglion cells, the most of glaucoma types is characterized by having a high intraocular pressure (IOP). This is the most important risk factor for this disease, even if it is not yet clear what are the pathogenic events connecting IOP to glaucoma phenotype. In any case the TM damage has a key role in the increasing of IOP.

BackgroundTrabecular meshwork and Schlemm's canal are the tissues appointed to modulate the aqueous humour outflow from the anterior chamber. The impairment of their functions drives to an intraocular pressure increase. The selective laser trabeculoplasty is a laser therapy of the trabecular meshwork able to decrease intraocular pressure. The exact response mechanism to this treatment has not been clearly delineated yet. The herein presented study is aimed at studying the gene expression changes induced in trabecular meshwork cells by selective laser trabeculoplasty (SLT) in order to better understand the mechanisms subtending its efficacy.Methodology/principal findingsPrimary human trabecular meshwork cells cultured in fibroblast medium underwent selective laser trabeculoplasty treatment. RNA was extracted from a pool of cells 30 minutes after treatment while the remaining cells were further cultured and RNA was extracted respectively 2 and 6 hours after treatment. Control cells stored in incubator in absence of SLT treatment were used as reference samples. Gene expression was evaluated by hybridization on miRNA-microarray and laser scanner analysis. Scanning electron microscopic examination was performed on 2 Trabecular meshwork samples after SLT at 4(th) and 6(th) hour from treatment. On the whole, selective laser trabeculoplasty modulates in trabecular meshwork the expression of genes involved in cell motility, intercellular connections, extracellular matrix production, protein repair, DNA repair, membrane repair, reactive oxygen species production, glutamate toxicity, antioxidant activities, and inflammation.Conclusions/significanceSLT did not induce any phenotypic alteration in TM samples. TM is a complex tissue possessing a great variety of function pivotal for the active regulation of aqueous humour outflow from the anterior chamber. SLT is able to modulate these functions at the postgenomic molecular level without inducing damage either at molecular or phenotypic levels.

This report describes a patient who had exaggerated responses to different inflammatory stimuli represented by laser and incisional surgery, respectively. These separate episodes should have a common link represented by a genetic predisposition to abnormal release of proinflammatory mediators within the eye. This 51-year old Hispanic woman showed a narrow iridocorneal angle with plateau iris configuration. Nd-YAG laser peripheral iridotomy was successfully performed to both eyes. No substantial changes in the iridotrabecular angle occurred despite patent iridotomies, thus confirming the diagnosis of plateau iris configuration. Argon laser iridoplasty was then performed to the right eye, while the left eye was scheduled for a later session. A severe inflammatory reaction within the anterior chamber developed after tapering of a one-week course of steroid therapy. Phacoemulsification of the lens was performed some months later when no signs of inflammation were detectable; no intraoperative complications occurred during surgery and an intraocular lens was placed. Cystoid macular edema developed four weeks after surgery despite no apparent risk factors, and resolved completely after anti-inflammatory medical therapy. Based on this case report, the unusual occurrence of severe iritis after laser treatment should be regarded as a risk factor for any other incisional or nonincisional procedures because it might indicate that the patient's ocular tissues are prone to release of abnormally elevated proinflammatory mediators. Although further studies are needed to confirm this predisposition, prophylactic adjunctive topical nonsteroidal anti-inflammatory drug administration after cataract surgery should be considered in such cases in order to prevent potentially sight-threatening conditions.

Objective To test the ability of dorzolamide hydrochloride and timolol maleate to display antioxidant effects. Methods Antioxidant activity was tested in whole trabecular meshwork (TM) tissue as collected from corneal donors' biopsy specimens, young (third passage) and old (10th passage) human TM cells, and acellular systems composed of pure DNA and subcellular fractions containing or devoid of mitochondria. Oxidative stress was induced by hydrogen peroxide. Monitored end points included DNA fragmentation as evaluated by the halo test, oxidative DNA damage in terms of 8-hydroxy-2′-deoxyguanosine, and mitochondrial function as evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide test. Results The antioxidant effect of dorzolamide and timolol were observed on TM biopsy specimens and human TM cells exposed to hydrogen peroxide. As evaluated in cell subfractions, timolol displays antioxidant activity regardless of mitochondria presence. Conversely, the antioxidant activity of dorzolamide was maximized in the presence of mitochondria-containing subcellular fractions and in young human TM cells with functional mitochondria. Conclusions The antioxidant effect of timolol was direct. The antioxidant effect of dorzolamide involves mitochondria and is likely to be exerted mainly during the early glaucoma phases when the mitochondrial damage in the TM tissue still occurs at low levels. Clinical Relevance Timolol has an antioxidant effect on the entire cell, whereas dorzolamide exerts protective activity toward oxidative stress only in the presence of intact mitochondria (ie, in endothelial cells that are younger when the cellular damage is still limited). The important role of mitochondrial damage in primary open- angle glaucoma is supported by the finding that mutant myocilin impairs mitochondrial functions in human TM meshwork cells.

As the only nourishment and scavenging source for most of the anterior and posterior chamber tissues in the eye, the aqueous humor represents one of the target for glaucoma. The aim of this study is to investigate the yet unexplored relationship between aqueous humor protein content and open-angle glaucoma (POAG) pathogenesis. Aqueous humor was collected from 10 POAG patients (cases) and 14 senile cataract patients (controls), matched for age and gender, undergoing surgery for trabeculectomy and cataract, respectively. Protein samples were cyanine-labeled and hybridized with antibody microarrays. Microarray signals were revealed by laser scanner, quantified, and compared by statistical analyses. Total protein amounts were not significantly different in patients versus controls. Conversely, a proteome cluster significantly modified in patients as compared to controls was identified as highly predictive for disease status. Selected proteins underwent dramatic variation, which was correlated to pathogenetic events characterizing POAG, including oxidative damage, mitochondrial damage, neural degeneration, and apoptosis. The results obtained indicate that proteomic analysis of aqueous humor is a new tool for POAG diagnosis in the case of otherwise uncertain disease recognition. Furthermore, this study allows a better understanding of mechanisms involved in the pathogenesis of POAG, the main cause of irreversible blindness worldwide.