Neuroprotective Effects of Transferrin in Experimental Glaucoma Models

Iron is essential for retinal metabolism, but an excess of ferrous iron causes oxidative stress. In glaucomatous eyes, retinal ganglion cell (RGC) death has been associated with dysregulation of iron homeostasis. Transferrin (TF) is an endogenous iron transporter that controls ocular iron levels. Intraocular administration of TF is neuroprotective in various models of retinal degeneration, preventing iron overload and reducing iron-induced oxidative stress. Herein, we assessed the protective effects of TF on RGC survival, using ex vivo rat retinal explants exposed to iron, NMDA-induced excitotoxicity, or CoCl₂-induced hypoxia, and an in vivo rat model of ocular hypertension (OHT). TF significantly preserved RGCs against FeSO₄-induced toxicity, NMDA-induced excitotoxicity, and CoCl₂-induced hypoxia. TF protected RGCs from apoptosis, ferroptosis, and necrosis. In OHT rats, TF reduced RGC loss by about 70% compared to vehicle-treated animals and preserved about 47% of the axons. Finally, increased iron staining was shown in the retina of a glaucoma patient’s eye as compared to non-glaucomatous eyes. These results indicate that TF can interfere with different cell-death mechanisms involved in glaucoma pathogenesis and demonstrate the ability of TF to protect RGCs exposed to elevated IOP. Altogether, these results suggest that TF is a promising treatment against glaucoma neuropathy.     

Risk Factors for Retinal Ganglion Cell Distress in Glaucoma and Neuroprotective Potential Intervention

Retinal ganglion cells (RGCs) are a population of neurons of the central nervous system (CNS) extending with their soma to the inner retina and with their axons to the optic nerve. Glaucoma represents a group of neurodegenerative diseases where the slow progressive death of RGCs results in a permanent loss of vision. To date, although Intra Ocular Pressure (IOP) is considered the main therapeutic target, the precise mechanisms by which RGCs die in glaucoma have not yet been clarified. In fact, Primary Open Angle Glaucoma (POAG), which is the most common glaucoma form, also occurs without elevated IOP. This present review provides a summary of some pathological conditions, i.e., axonal transport blockade, glutamate excitotoxicity and changes in pro-inflammatory cytokines along the RGC projection, all involved in the glaucoma cascade. Moreover, neuro-protective therapeutic approaches, which aim to improve RGC degeneration, have also been taken into consideration.     

Neuroprotective Effect of Kinase Inhibition in Ischemic Factor Modeling In Vitro

The contribution of many neuronal kinases to the adaptation of nerve cells to ischemic damage and their effect on functional neural network activity has not yet been studied. The aim of this work is to study the role of the four kinases belonging to different metabolic cascades (SRC, Ikkb, eEF2K, and FLT4) in the adaptive potential of the neuron-glial network for modeling the key factors of ischemic damage. We carried out a comprehensive study on the effects of kinases blockade on the viability and network functional calcium activity of nerve cells under ischemic factor modeling in vitro. Ischemic factor modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). The most significant neuroprotective effect was shown in the blockade of FLT4 kinase in the simulation of hypoxia. The studies performed revealed the role of FLT4 in the development of functional dysfunction in cerebrovascular accidents and created new opportunities for the study of this enzyme and its blockers in the formation of new therapeutic strategies.     

Neuroprotective Effect of Statins in a Rat Model of Chronic Ocular Hypertension

Glaucoma is an optic neuropathy in which the degeneration of retinal ganglion cells (RGCs) results in irreversible vison loss. Therefore, neuroprotection of RGCs from glaucomatous afflictions is crucial for glaucoma treatment. In this study, we aimed to investigate the beneficial effects of statins in the protection of RGCs using a rat model. Glaucomatous injury was induced in rats by chronic ocular hypertension (OHT) achieved after performing a circumlimbal suture. The rats were given either statins such as simvastatin and atorvastatin or a solvent weekly for 6 weeks. Retina sections underwent hematoxylin and eosin, Brn3a, or cleaved casepase-3 staining to evaluate RGC survival. In addition, modulation of glial activation was assessed. While the retinas without statin treatment exhibited increased RGC death due to chronic OHT, statins promoted the survival of RGCs and reduced apoptosis. Statins also suppressed chronic OHT-mediated glial activation in the retina. Our results demonstrate that statins exert neuroprotective effects in rat retinas exposed to chronic OHT, which may support the prospect of statins being a glaucoma treatment.     

Inhibition of Neuronal Necroptosis Mediated by RIPK1 Provides Neuroprotective Effects on Hypoxia and Ischemia In Vitro and In Vivo

Ischemic brain injury is a widespread pathological condition, the main components of which are a deficiency of oxygen and energy substrates. In recent years, a number of new forms of cell death, including necroptosis, have been described. In necroptosis, a cascade of interactions between the kinases RIPK1 and RIPK3 and the MLKL protein leads to the formation of a specialized death complex called the necrosome, which triggers MLKL-mediated destruction of the cell membrane and necroptotic cell death. Necroptosis probably plays an important role in the development of ischemia/reperfusion injury and can be considered as a potential target for finding methods to correct the disruption of neural networks in ischemic damage. In the present study, we demonstrated that blockade of RIPK1 kinase by Necrostatin-1 preserved the viability of cells in primary hippocampal cultures in an in vitro model of glucose deprivation. The effect of RIPK1 blockade on the bioelectrical and metabolic calcium activity of neuron-glial networks in vitro using calcium imaging and multi-electrode arrays was assessed for the first time. RIPK1 blockade was shown to partially preserve both calcium and bioelectric activity of neuron-glial networks under ischemic factors. However, it should be noted that RIPK1 blockade does not preserve the network parameters of the collective calcium dynamics of neuron-glial networks, despite the maintenance of network bioelectrical activity (the number of bursts and the number of spikes in the bursts). To confirm the data obtained in vitro, we studied the effect of RIPK1 blockade on the resistance of small laboratory animals to in vivo modeling of hypoxia and cerebral ischemia. The use of Necrostatin-1 increases the survival rate of C57BL mice in modeling both acute hypobaric hypoxia and ischemic brain damage.     

Neuroprotection,Growth Factors and BDNF-TrkB Signalling in Retinal Degeneration

Neurotrophic factors play key roles in the development and survival of neurons. The potent neuroprotective effects of neurotrophic factors, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-line derived neurotrophic factor (GDNF) and nerve growth factor (NGF), suggest that they are good therapeutic candidates for neurodegenerative diseases. Glaucoma is a neurodegenerative disease of the eye that causes irreversible blindness. It is characterized by damage to the optic nerve, usually due to high intraocular pressure (IOP), and progressive degeneration of retinal neurons called retinal ganglion cells (RGCs). Current therapy for glaucoma focuses on reduction of IOP, but neuroprotection may also be beneficial. BDNF is a powerful neuroprotective agent especially for RGCs. Exogenous application of BDNF to the retina and increased BDNF expression in retinal neurons using viral vector systems are both effective in protecting RGCs from damage. Furthermore, induction of BDNF expression by agents such as valproic acid has also been beneficial in promoting RGC survival. In this review, we discuss the therapeutic potential of neurotrophic factors in retinal diseases and focus on the differential roles of glial and neuronal TrkB in neuroprotection. We also discuss the role of neurotrophic factors in neuroregeneration.     

Opa1 Deficiency Promotes Development of Retinal Vascular Lesions in Diabetic Retinopathy

This study investigates whether reduced optic atrophy 1 (Opa1) level promotes apoptosis and retinal vascular lesions associated with diabetic retinopathy (DR). Four groups of mice: wild type (WT) control mice, streptozotocin (STZ)-induced diabetic mice, Opa1^+/− mice, and diabetic Opa1^+/− mice were used in this study. 16 weeks after diabetes onset, retinas were assessed for Opa1 and Bax levels by Western blot analysis, and retinal networks were examined for acellular capillaries (AC) and pericyte loss (PL). Apoptotic cells were detected in retinal capillaries using TUNEL assay, and caspase-3 activity was assessed using fluorometric analysis. Opa1 expression was significantly downregulated in retinas of diabetic and Opa1^+/− mice compared with those of WT mice. Inducing diabetes further decreased Opa1 expression in retinas of Opa1^+/− mice. Increased cytochrome c release concomitant with increased level of pro-apoptotic Bax and elevated caspase-3 activity were observed in retinas of diabetic and Opa1^+/− mice; the number of TUNEL-positive cells and AC/PL was also significantly increased. An additional decrease in the Opa1 level in retinas of diabetic Opa1^+/− mice exacerbated the development of apoptotic cells and AC/PL compared with those of diabetic mice. Diabetes-induced Opa1 downregulation contributes, at least in part, to the development of retinal vascular lesions characteristic of DR.     

The Role of Inflammation in Retinal Neurodegeneration and Degenerative Diseases

Retinal neurodegeneration is predominantly reported as the apoptosis or impaired function of the photoreceptors. Retinal degeneration is a major causative factor of irreversible vision loss leading to blindness. In recent years, retinal degenerative diseases have been investigated and many genes and genetic defects have been elucidated by many of the causative factors. An enormous amount of research has been performed to determine the pathogenesis of retinal degenerative conditions and to formulate the treatment modalities that are the critical requirements in this current scenario. Encouraging results have been obtained using gene therapy. We provide a narrative review of the various studies performed to date on the role of inflammation in human retinal degenerative diseases such as age-related macular degeneration, inherited retinal dystrophies, retinitis pigmentosa, Stargardt macular dystrophy, and Leber congenital amaurosis. In addition, we have highlighted the pivotal role of various inflammatory mechanisms in the progress of retinal degeneration. This review also offers an assessment of various therapeutic approaches, including gene-therapies and stem-cell-based therapies, for degenerative retinal diseases.     

Tools and Biomarkers for the Study of Retinal Ganglion Cell Degeneration

The retina is part of the central nervous system, its analysis may provide an idea of the health and functionality, not only of the retina, but also of the entire central nervous system, as has been shown in Alzheimer’s or Parkinson’s diseases. Within the retina, the ganglion cells (RGC) are the neurons in charge of processing and sending light information to higher brain centers. Diverse insults and pathological states cause degeneration of RGC, leading to irreversible blindness or impaired vision. RGCs are the measurable endpoints in current research into experimental therapies and diagnosis in multiple ocular pathologies, like glaucoma. RGC subtype classifications are based on morphological, functional, genetical, and immunohistochemical aspects. Although great efforts are being made, there is still no classification accepted by consensus. Moreover, it has been observed that each RGC subtype has a different susceptibility to injury. Characterizing these subtypes together with cell death pathway identification will help to understand the degenerative process in the different injury and pathological models, and therefore prevent it. Here we review the known RGC subtypes, as well as the diagnostic techniques, probes, and biomarkers for programmed and unprogrammed cell death in RGC.     

Damage-Associated Molecular Patterns (DAMPs) in Retinal Disorders

Damage-associated molecular patterns (DAMPs) are endogenous danger molecules released from the extracellular and intracellular space of damaged tissue or dead cells. Recent evidence indicates that DAMPs are associated with the sterile inflammation caused by aging, increased ocular pressure, high glucose, oxidative stress, ischemia, mechanical trauma, stress, or environmental conditions, in retinal diseases. DAMPs activate the innate immune system, suggesting their role to be protective, but may promote pathological inflammation and angiogenesis in response to the chronic insult or injury. DAMPs are recognized by specialized innate immune receptors, such as receptors for advanced glycation end products (RAGE), toll-like receptors (TLRs) and the NOD-like receptor family (NLRs), and purine receptor 7 (P2X7), in systemic diseases. However, studies describing the role of DAMPs in retinal disorders are meager. Here, we extensively reviewed the role of DAMPs in retinal disorders, including endophthalmitis, uveitis, glaucoma, ocular cancer, ischemic retinopathies, diabetic retinopathy, age-related macular degeneration, rhegmatogenous retinal detachment, proliferative vitreoretinopathy, and inherited retinal disorders. Finally, we discussed DAMPs as biomarkers, therapeutic targets, and therapeutic agents for retinal disorders.     

Influence of Umbelliferone on the Anticonvulsant and Neuroprotective Activity of Selected Antiepileptic Drugs: An In Vivo and In Vitro Study

Umbelliferone (7-hydroxycoumarin; UMB) is a coumarin with many biological properties, including antiepileptic activity. This study evaluated the effect of UMB on the ability of classical and novel antiepileptic drugs (e.g., lacosamide (LCM), levetiracetam (LEV), phenobarbital (PB) and valproate (VPA)) to prevent seizures evoked by the 6-Hz corneal-stimulation-induced seizure model. The study also evaluated the influence of this coumarin on the neuroprotective properties of these drugs in two in vitro models of neurodegeneration, including trophic stress and excitotoxicity. The results indicate that UMB (100 mg/kg, i.p.) significantly enhanced the anticonvulsant action of PB (p < 0.01) and VPA (p < 0.05), but not that of LCM orLEV, in the 6-Hz test. Whether alone or in combination with other anticonvulsant drugs (at their ED₅₀ values from the 6-Hz test), UMB (100 mg/kg) did not affect motor coordination; skeletal muscular strength and long-term memory, as determined in the chimney; grip strength; or passive avoidance tests, respectively. Pharmacokinetic characterization revealed that UMB had no impact on total brain concentrations of PB or VPA in mice. The in vitro study indicated that UMB has neuroprotective properties. Administration of UMB (1 µg/mL), together with antiepileptic drugs, mitigated their negative impact on neuronal viability. Under trophic stress (serum deprivation) conditions, UMB enhanced the neurotrophic abilities of all the drugs used. Moreover, this coumarin statistically enhanced the neuroprotective effects of PB (p < 0.05) and VPA (p < 0.001) in the excitotoxicity model of neurodegeneration. The obtained results clearly indicate a positive effect of UMB on the anticonvulsant and neuroprotective properties of the selected drugs.     

Antioxidant Drug Therapy Approaches for Neuroprotection in Chronic Diseases of the Retina

The molecular pathways contributing to visual signal transduction in the retina generate a high energy demand that has functional and structural consequences such as vascularization and high metabolic rates contributing to oxidative stress. Multiple signaling cascades are involved to actively regulate the redox state of the retina. Age-related processes increase the oxidative load, resulting in chronically elevated levels of oxidative stress and reactive oxygen species, which in the retina ultimately result in pathologies such as glaucoma or age-related macular degeneration, as well as the neuropathic complications of diabetes in the eye. Specifically, oxidative stress results in deleterious changes to the retina through dysregulation of its intracellular physiology, ultimately leading to neurodegenerative and potentially also vascular dysfunction. Herein we will review the evidence for oxidative stress-induced contributions to each of the three major ocular pathologies, glaucoma, age-related macular degeneration, and diabetic retinopathy. The premise for neuroprotective strategies for these ocular disorders will be discussed in the context of recent clinical and preclinical research pursuing novel therapy development approaches.     

Sigma-1 Receptor in Retina: Neuroprotective Effects and Potential Mechanisms

Retinal degenerative diseases are the major factors leading to severe visual impairment and even irreversible blindness worldwide. The therapeutic approach for retinal degenerative diseases is one extremely urgent and hot spot in science research. The sigma-1 receptor is a novel, multifunctional ligand-mediated molecular chaperone residing in endoplasmic reticulum (ER) membranes and the ER-associated mitochondrial membrane (ER-MAM); it is widely distributed in numerous organs and tissues of various species, providing protective effects on a variety of degenerative diseases. Over three decades, considerable research has manifested the neuroprotective function of sigma-1 receptor in the retina and has attempted to explore the molecular mechanism of action. In the present review, we will discuss neuroprotective effects of the sigma-1 receptor in retinal degenerative diseases, mainly in aspects of the following: the localization in different types of retinal neurons, the interactions of sigma-1 receptors with other molecules, the correlated signaling pathways, the influence of sigma-1 receptors to cellular functions, and the potential therapeutic effects on retinal degenerative diseases.     

The Role of Autophagy in Chemical Proteasome Inhibition Model of Retinal Degeneration

We recently demonstrated that chemical proteasome inhibition induced inner retinal degeneration, supporting the pivotal roles of the ubiquitin–proteasome system in retinal structural integrity maintenance. In this study, using beclin1-heterozygous (Becn1-Het) mice with autophagic dysfunction, we tested our hypothesis that autophagy could be a compensatory retinal protective mechanism for proteasomal impairment. Despite the reduced number of autophagosome, the ocular tissue morphology and intraocular pressure were normal. Surprisingly, Becn1-Het mice experienced the same extent of retinal degeneration as was observed in wild-type mice, following an intravitreal injection of a chemical proteasome inhibitor. Similarly, these mice equally responded to other chemical insults, including endoplasmic reticulum stress inducer, N-methyl-D-aspartate, and lipopolysaccharide. Interestingly, in cultured neuroblastoma cells, we found that the mammalian target of rapamycin-independent autophagy activators, lithium chloride and rilmenidine, rescued these cells against proteasome inhibition-induced death. These results suggest that Becn1-mediated autophagy is not an effective intrinsic protective mechanism for retinal damage induced by insults, including impaired proteasomal activity; furthermore, autophagic activation beyond normal levels is required to alleviate the cytotoxic effect of proteasomal inhibition. Further studies are underway to delineate the precise roles of different forms of autophagy, and investigate the effects of their activation in rescuing retinal neurons under various pathological conditions.     

Tonabersat Inhibits Connexin43 Hemichannel Opening and Inflammasome Activation in an In Vitro Retinal Epithelial Cell Model of Diabetic Retinopathy

This study was undertaken to evaluate the connexin hemichannel blocker tonabersat for the inhibition of inflammasome activation and use as a potential treatment for diabetic retinopathy. Human retinal pigment epithelial cells (ARPE-19) were stimulated with hyperglycemia and the inflammatory cytokines IL-1β and TNFα in order to mimic diabetic retinopathy molecular signs in vitro. Immunohistochemistry was used to evaluate the effect of tonabersat treatment on NLRP3, NLRP1, and cleaved caspase-1 expression and distribution. A Luminex cytokine release assay was performed to determine whether tonabersat affected proinflammatory cytokine release. NLRP1 was not activated in ARPE-19 cells, and IL-18 was not produced under disease conditions. However, NLRP3 and cleaved caspase-1 complex formation increased with hyperglycemia and cytokine challenge but was inhibited by tonabersat treatment. It also prevented the release of proinflammatory cytokines IL-1β, VEGF, and IL-6. Tonabersat therefore has the potential to reduce inflammasome-mediated inflammation in diabetic retinopathy.     

Neuroprotective Effects of Pre-Treament with l-Carnitine and Acetyl-l-Carnitine on Ischemic Injury In Vivo and In Vitro

The therapeutic effect of stroke is hampered by the lack of neuroprotective drugs against ischemic insults beyond the acute phase. Carnitine plays important roles in mitochondrial metabolism and in modulating the ratio of coenzyme A (CoA)/acyl-CoA. Here, we investigate the neuroprotective effects of l-carnitine (LC) and Acetyl-l-carnitine (ALC) pre-treatment on ischemic insults under the same experimental conditions. We used a transient middle cerebral artery occlusion (MCAO) model to evaluate the protective roles of LC and ALC in acute focal cerebral ischemia in vivo and to understand the possible mechanisms using model of PC12 cell cultures in vitro. Results showed that ALC, but not LC, decreased infarction size in SD rats after MCAO in vivo. However, both LC and ALC pretreatment reduced oxygen-glucose deprivation (OGD)-induced cell injury and decreased OGD-induced cell apoptosis and death in vitro; at the same time, both of them increased the activities of super oxide dismutase (SOD) and ATPase, and decreased the concentration of malondialdehyde (MDA) in vitro. Thus, our findings suggested that LC and ALC pre-treatment are highly effective in the prevention of neuronal cell against ischemic injury in vitro, however, only ALC has the protective effect on neuronal cell injury after ischemia in vivo.     

Nucleobase-Derived Nitrones: Synthesis and Antioxidant and Neuroprotective Activities in an In Vitro Model of Ischemia–Reperfusion

Herein, we report the synthesis, antioxidant, and neuroprotective properties of some nucleobase-derived nitrones named 9a–i. The neuroprotective properties of nitrones, 9a–i, were measured against an oxygen-glucose-deprivation in vitro ischemia model using human neuroblastoma SH-SY5Y cells. Our results indicate that nitrones, 9a–i, have better neuroprotective and antioxidant properties than α-phenyl-N-tert-butylnitrone (PBN) and are similar to N-acetyl-L-cysteine (NAC), a well-known antioxidant and neuroprotective agent. The nitrones with the highest neuroprotective capacity were those containing purine nucleobases (nitrones 9f, g, B = adenine, theophylline), followed by nitrones with pyrimidine nucleobases with H or F substituents at the C5 position (nitrones 9a, c). All of these possess EC₅₀ values in the range of 1–6 μM and maximal activities higher than 100%. However, the introduction of a methyl substituent (nitrone 9b, B = thymine) or hard halogen substituents such as Br and Cl (nitrones 9d, e, B = 5-Br and 5-Cl uracil, respectively) worsens the neuroprotective activity of the nitrone with uracil as the nucleobase (9a). The effects on overall metabolic cell capacity were confirmed by results on the high anti-necrotic (EC_50′s ≈ 2–4 μM) and antioxidant (EC_50′s ≈ 0.4–3.5 μM) activities of these compounds on superoxide radical production. In general, all tested nitrones were excellent inhibitors of superoxide radical production in cultured neuroblastoma cells, as well as potent hydroxyl radical scavengers that inhibit in vitro lipid peroxidation, particularly, 9c, f, g, presenting the highest lipoxygenase inhibitory activity among the tested nitrones. Finally, the introduction of two nitrone groups at 9a and 9d (bis-nitronas 9g, i) did not show better neuroprotective effects than their precursor mono-nitrones. These results led us to propose nitrones containing purine (9f, g) and pyrimidine (9a, c) nucleobases as potential therapeutic agents for the treatment of cerebral ischemia and/or neurodegenerative diseases, leading us to further investigate their effects using in vivo models of these pathologies.     

Anti-Inflammatory Effects of GLP-1R Activation in the Retina

Glucagon-like peptide-1 (GLP-1) is an incretin hormone, mainly produced by enteroendocrine L cells, which participates in the regulation of glucose homeostasis, and in reduction in body weight by promoting satiety. Actions of GLP-1 are mediated by activation of its receptor GLP-1R, which is widely expressed in several tissues including the retina. The effects of GLP-1R activation are useful in the management of type 2 diabetes mellitus (T2DM). In addition, the activation of GLP-1R has anti-inflammatory effects in several organs, suggesting that it may be also useful in the treatment of inflammatory diseases. Inflammation is a common element in the pathogenesis of several ocular diseases, and the protective effects of treatment with GLP-1 emerged also in retinal diseases. In this review we highlight the anti-inflammatory effects of GLP-1R activation in the retina. Firstly, we summarized the pathogenic role of inflammation in ocular diseases. Then, we described the pleiotropic effects of GLP-1R activation on the cellular components of the retina which are mainly involved in the pathogenesis of inflammatory retinal diseases: the retinal ganglion cells, retinal pigment epithelial cells and endothelial cells.