Immunocytochemical Analysis of Crocin against Oxidative Stress in Trigeminal Sensory Neurons Innervating the Cornea.

Corneal diseases are a major cause of vision loss, often associated with aging, trauma and disease. Damage to corneal sensory innervation leads to discomfort and pain. Environmental stressors, such as short-wavelength light, can induce oxidative stress that alters mitochondrial function and affects cell and tissue homeostasis, including corneal innervation. Cellular antioxidant mechanisms may attenuate oxidative stress. This study investigates crocin, a derivative of saffron, as a potential antioxidant therapy. In vitro rat trigeminal sensory ganglion neurons were exposed to both sodium azide and blue light overexposure as a model of oxidative damage. Crocin was used as a neuroprotective agent. Mitochondrial and cytoskeletal markers were studied by immunofluorescence analysis to determine oxidative damage and neuroprotection. In vivo corneal innervation degeneration was evaluated in cornea whole mount preparations using Sholl analyses. Blue light exposure induces oxidative stress that affects trigeminal neuron mitochondria and alters sensory axon dynamics in vitro, and it also affects corneal sensory innervation in an in vivo model. Our results show that crocin was effective in preserving mitochondrial function and protecting corneal sensory neurons from oxidative stress. Crocin appears to be a promising candidate for the neuroprotection of corneal innervation.

PRGF Membrane with Tailored Optical Properties Preserves the Cytoprotective Effect of Plasma Rich in Growth Factors: In Vitro Model of Retinal Pigment Epithelial Cells.

The present study evaluates the ability of a novel plasma rich in growth factors (PRGF) membrane with improved optical properties to reduce oxidative stress in retinal pigment epithelial cells (ARPE-19 cells) exposed to blue light. PRGF was obtained from three healthy donors and divided into four main groups: (i) PRGF membrane (M-PRGF), (ii) PRGF supernatant (S-PRGF), (iii) platelet-poor plasma (PPP) membrane diluted 50% with S-PRGF (M-PPP 50%), and (iv) M-PPP 50% supernatant (S-PPP 50%). ARPE-19 cells were exposed to blue light and then incubated with the different PRGF-derived formulations or control for 24 and 48 h under blue light exposure. Mitochondrial and cell viability, reactive oxygen species (ROS) production, and heme oxygenase-1 (HO-1) and ZO-1 expression were evaluated. Mitochondrial viability and cell survival were significantly increased after treatment with the different PRGF-derived formulations. ROS synthesis and HO-1 expression were significantly reduced after cell treatment with any of the PRGF-derived formulations. Furthermore, the different PRGF-derived formulations significantly increased ZO-1 expression in ARPE-19 exposed to blue light. The new PRGF membrane with improved optical properties and its supernatant (M-PPP 50% and S-PPP 50%) protected and reversed blue light-induced oxidative stress in ARPE-19 cells at levels like those of a natural PRGF membrane and its supernatant.

Synthetic Heparan Sulfate Mimetic Polymer Enhances Corneal Nerve Regeneration and Wound Healing after Experimental Laser Ablation Injury in Mice.

(1) Background: Abnormal corneal wound healing compromises visual acuity and can lead to neuropathic pain. Conventional treatments usually fail to restore the injured corneal tissue. In this study, we evaluated the effectiveness of a synthetic heparan sulfate mimetic polymer (HSmP) in a mouse model of corneal wound healing. (2) Methods: A surgical laser ablation affecting the central cornea and subbasal nerve plexus of mice was used as a model of the wound-healing assay. Topical treatment with HSmP was contrasted to its vehicle and a negative control (BSS). Corneal repair was studied using immunofluorescence to cell proliferation (Ki67), apoptosis (TUNEL assay), myofibroblast transformation (αSMA), assembly of epithelial cells (E-cadherin) and nerve regeneration (ß-tubulin III). (3) Results: At the end of the treatment, normal epithelial cytoarchitecture and corneal thickness were achieved in HSmP-treated animals. HSmP treatment reduced myofibroblast occurrence compared to eyes irrigated with vehicle (p < 0.01) or BSS (p < 0.001). The HSmP group showed 50% more intraepithelial nerves than the BSS or vehicle groups. Only HSmP-treated corneas improved the visual quality to near transparent. (4) Conclusions: These results suggest that HSmP facilitates the regeneration of the corneal epithelium and innervation, as well as restoring transparency and reducing myofibroblast scarring after laser experimental injury.

Human Stem Cell Transplantation for Retinal Degenerative Diseases: Where Are We Now?

Background and Objectives: Irreversible visual impairment is mainly caused by retinal degenerative diseases such as age-related macular degeneration and retinitis pigmentosa. Stem cell research has experienced rapid progress in recent years, and researchers and clinical ophthalmologists are trying to implement this promising technology to treat retinal degeneration. The objective of this systematic review is to analyze currently available data from clinical trials applying stem cells to treat human retinal diseases. Materials and Methods: We performed a systematic literature search in PubMed to identify articles related with stem cell therapies to retinal diseases published prior to September 2021. Furthermore, a systematic search in ClinicalTrials (NIH U.S. National Library of Medicine) was performed to identify clinical trials using stem cells to treat retinal diseases. A descriptive analysis of status, conditions, phases, interventions, and outcomes is presented here. Conclusions: To date, no available therapy based on stem cell transplantation is approved for use with patients. However, numerous clinical trials are currently finishing their initial phases and, in general, the outcomes related to implantation techniques and their long-term safety seem promising. In the next few years, we expect to see quantifiable results pertaining to visual function improvement.

Protector Role of Intraocular Lenses under Artificial Light Conditions.

INTRODUCTION: The aim of this work was to analyze, in an in vitro model, the possible protective effects of ultraviolet- (UV-) or UV/blue-filtering intraocular lens (IOL) under light-emitting diode (LED) lighting conditions. METHODS: Ten models of IOLs were evaluated. Light transmission spectrum was recorded from 300 to 800 nm, in steps of 1 nm. Photodamage in vitro model was induced in ARPE-19 cells by blue LED light (465-475 nm). Changes in cell viability and oxidative stress variables were studied to assess the protective effect of IOLs. RESULTS: UV/blue-filtering IOLs models block blue light spectrum in different proportion and UV-filtering IOLs blocking wavelength below 400 nm. However, in vitro study under blue LED light exposure does not show protective effects related with mitochondrial dysfunction and oxidative stress of UV/blue-filtering IOLs. CONCLUSIONS: The current in vitro study suggests that UV/blue filtering IOLs are not useful in terms of photoprotection in artificial light conditions. The results obtained indicate that it is needed to give attention to other IOL parameters besides the type of filter, as it seems they could have influence on the protective role.

Plasma Rich in Growth Factors Promotes Autophagy in ARPE19 Cells in Response to Oxidative Stress Induced by Blue Light.

Age-related macular degeneration (AMD) causes the degeneration of photoreceptors and retinal cells leading to vision loss in older subjects. Among possible exogenous risk factors, it has been recently proposed that long-term exposure to blue light could aggravate the course of AMD. In the search for therapeutic options, plasma rich in growth factors (PRGF) has been shown to enhance cell antioxidant pathways and protect photoreceptors against the harm produced by blue light, although its mechanism of action remains unknown. One possible mechanism, autophagy, is one of the most conservative cell renewal systems used in eukaryotes to destroy cellular components that have been damaged by some kind of insult. The oxidative stress of exposure to blue light is known to induce cell autophagy. In this study, we examined the combined effects on autophagy of blue light and PRGF in a retinal cell line, ARPE19. In response to treatment with both PRGF and blue light, we detected the modulated expression of autophagy markers such as NF-kB, p62/sqstm1, Atg5, LC3 and Beclin1, and inflammatory markers such as IL1B and IL18. Our findings suggest that PRGF promotes cell autophagy in response to exposure to blue light.

Plasma Rich in Growth Factors Enhances Cell Survival after in Situ Retinal Degeneration.

PURPOSE: The purpose of this study was to examine the effect of plasma rich in growth factors (PRGFs) under blue light conditions in an in vivo model of retinal degeneration. METHODS: Male Wistar rats were exposed to dark/blue light conditions for 9 days. On day 7, right eyes were injected with saline and left eyes with PRGF. Electroretinography (ERG) and intraocular pressure (IoP) measurements were performed before and after the experiment. After sacrifice, retinal samples were collected. Hematoxylin and eosin staining was performed to analyze the structure of retinal sections. Immunofluorescence for brain-specific homeobox/POU domain protein 3A (Brn3a), choline acetyltransferase (ChAT), rhodopsin, heme oxygenase-1 (HO-1), and glial fibrillary acidic protein (GFAP) was performed to study the retinal conditions. RESULTS: Retinal signaling measured by ERG was reduced by blue light and recovered with PRGF; however, IoP measurements did not show significant differences among treatments. Blue light reduced the expression for Brn3a, ChAT, and rhodopsin. Treatment with PRGF showed a recovery in their expressions. HO-1 and GFAP results showed that blue light increased their expression but the use of PRGF reduced the effect of light. CONCLUSIONS: Blue light causes retinal degeneration. PRGF mitigated the injury, restoring the functionality of these cells and maintaining the tissue integrity.

Plasma rich in growth factors reduces blue light-induced oxidative damage on retinal pigment epithelial cells and restores their homeostasis by modulating vascular endothelial growth factor and pigment epithelium-derived factor expression.

BACKGROUND: This study analysed the effectiveness of plasma rich in growth factors (PRGF) in reducing the oxidative stress induced by blue light exposition on retinal pigment epithelial (RPE) cells. METHODS: Blood from six healthy donors was collected to obtain the PRGF. Retinal pigment epithelium (ARPE-19) cells were exposed to blue light. Then, cells were incubated with PRGF or with control for 24 and 48 hours maintaining exposure to blue light. The cytoprotective effect of PRGF on ARPE cells was evaluated by measuring the cell viability, the reactive oxygen species (ROS) production and the expression of different proteins such as heme oxygenase 1 (HO-1), catalase (CAT), superoxide dismutase (SOD-1), apoptosis-inducing factor (AIF), pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF). RESULTS: The cell viability increased significantly at 24 and 48 hours after PRGF treatment compared to the control group. ROS synthesis was significantly reduced in PRGF-treated cells with respect to control. Furthermore, the levels of HO-1, SOD-1 and AIF were significantly reduced after PRGF treatment at both times of treatment. However, CAT levels were only significantly reduced after PRGF treatment at 48 hours. The high expression of VEGF by RPE cells exposed to blue light was only counterbalanced in the PRGF group by increasing the expression of PEDF in comparison to the control group. CONCLUSION: The present results show that PRGF treatment reduces the cytotoxic effects induced in RPE cells exposed to an oxidative stress environment. Furthermore, PRGF treatment preserves the mitochondrial activity and cell viability of RPE cells subjected to an oxidative stress.

Antioxidant Role of PRGF on RPE Cells after Blue Light Insult as a Therapy for Neurodegenerative Diseases.

Oxidative stress has a strong impact on the development of retinal diseases such as age-related macular degeneration (AMD). Plasma rich in growth factors (PRGF) is a novel therapeutic approach in ophthalmological pathologies. The aim of this study was to analyze the antioxidant effect of PRGF in retinal epithelial cells (EPR) in in vitro and ex vivo retinal phototoxicity models. In vitro analyses were performed on ARPE19 human cell line. Viability and mitochondrial status were assessed in order to test the primary effects of PRGF. GSH level, and protein and gene expression of the main antioxidant pathway (Keap1, Nrf2, GCL, HO-1, and NQO1) were also studied. Ex vivo analyses were performed on rat RPE, and HO-1 and Nrf2 gene and protein expression were evaluated. The results show that PRGF reduces light insult by stimulating the cell response against oxidative damage and modulates the antioxidant pathway. We conclude that PRGF's protective effect could prove useful as a new therapy for treating neurodegenerative disorders such as AMD.

Blue light negatively affects the survival of ARPE19 cells through an action on their mitochondria and blunted by red light.

PURPOSE: To ascertain whether red light, known to enhance mitochondrial function, can blunt a blue light insult to ARPE19 cells in culture. METHODS: Semi-confluent ARPE19 cells cultured in 10% FBS were subjected to various regimes of treatment with blue (465-475 nm, 800 lux, 26 W/m2 ) and red (625-635 nm, 950 lux, 6.5 W/m2 ) light, as well as with toxins that inactivate specific enzymes associated with mitochondrial oxidative phosphorylation. Cultures were then analysed for cell viability (MTT assay), mitochondrial status (JC-1), ROS formation, immunocytochemistry and the activation of specific proteins by electrophoresis/Western blotting. In addition, ARPE19 cells were cultured in polycarbonate membrane inserts in culture medium containing 1% FBS. Such cultures were exposed to cycles of red, blue or a combination of red and blue light for up to 6 weeks. Culture medium was changed and the trans-epithelium membrane resistance (TER) of the inserts-containing cells was measured twice weekly. RESULTS: ARPE19 cells in culture are affected negatively when exposed to blue light. This is indicated by a loss of viability, a depolarization of their mitochondria and a stimulation of ROS. Moreover, blue light causes an up-regulation of HO-1 and phospho-p-38-MAPK and a cleavage of apoptosis inhibitory factor, proteins which are all known to be activated during cell death. All of these negative effects of blue light are significantly blunted by the red light administered after the blue light insult in each case. ARPE19 cell loss of viability and mitochondrial potential caused by toxins that inhibit specific mitochondrial enzyme complexes was additive to an insult delivered by blue light in each case. After a time, ARPE19 cells in culture express the tight junction protein ZO-1, which is affected by blue light. The development of tight junctions between ARPE19 cells grown in inserts reached a steady peak of resistance after about 40 days and then increased very slightly over the next 40 days when still in darkness. However, maximum resistance was significantly attenuated, when cultures were treated with cycles of blue light after the initial 40 days in the dark and counteracted significantly when the blue light cycle insult was combined with red light. CONCLUSION: Blue light affects mitochondrial function and also the development tight junctions between ARPE19 cells, which results in a loss of cell viability. Importantly, red light delivered after a blue light insult is significantly blunted. These findings argue for the therapeutic use of red light as a noninvasive procedure to attenuate insults caused by blue light and other insults to retinal pigment epithelial cell mitochondria that are likely to occur in age-related macular degeneration.

Near infra-red light attenuates corneal endothelial cell dysfunction in situ and in vitro.

In the present study mechanical damage to the corneal endothelium was induced by elevation of intraocular pressure (IOP, 140 mmHg, 60 min) to one eye of rats, delivered either in complete darkness or in the presence of red light (16.5 W/m2, 3000 lx, 625-635 nm). IOP raised in the dark revealed the endothelium to be damaged as staining for the gap junction protein ZO-1 was irregular in appearance with some cells displaced in position or lost to leave gaps or holes. This damage was clearly attenuated when red light was focused through the pupil during the insult of raised IOP. Moreover, staining of endothelium with JC-1 dye showed mitochondria to be activated by both elevated IOP and red light but the activation of mitochondria persisted longer for red light. We interpret this finding to suggest that raised IOP causes apoptosis of endothelial cells and that their mitochondria are activated in the initial stages of the process. In contrast, red light activates mitochondria to induce a protective mechanism to counteract the negative influence of raised IOP on endothelial cells. Evidence is provided to support this notion by the finding that red light stimulates mitochondrial cytochrome oxidase IV (COX IV). Moreover, mitochondria in corneal endothelial cell cultures are activated by red light, revealed by staining with JC-1, that results in an increased rate of proliferation and are also able to counteract toxic insults (sodium azide or cobalt chloride) to the cultures. The present studies therefore show that a non-toxic level of red light attenuates damage to the corneal endothelium both in situ and in vitro through action on COX IV located in mitochondria that results in an enhancement of a cell's survival mechanisms. The study provides proof of principle for the non-invasive use of red-light therapy to attenuate any dysfunctions associated with the corneal endothelium and so preserve maximum visual acuity.

Visual light effects on mitochondria: The potential implications in relation to glaucoma.

Light of different wave-lengths have the potential to interact with four major mitochondrial protein complexes that are involved in the generation of ATP. Neurones of the central nervous system have an absolute dependence on mitochondrial generated ATP. Laboratory studies show that short-wave or blue light (400-480nm) that impinges on the retina affect flavin and cytochrome constituents associated with mitochondria to decrease the rate of ATP formation, stimulate ROS and results in cell death. This suggests that blue light could potentially have a negative influence on retinal ganglion cell (RGC) mitochondria that are abundant and not shielded by macular pigments as occurs for photoreceptor mitochondria. This might be of significance in glaucoma where it is likely that RGC mitochondria are already affected and therefore be more susceptible to blue light. Thus simply filtering out some natural blue light from entering the eye might be beneficial for the treatment of glaucoma. Long-wave or red light (650-800nm) affects mitochondrial complex IV or cytochrome oxidase to increase the rate of formation of ATP and ROS causing the generation of a number of beneficial factors. Significantly, laboratory studies show that increasing the normal amount of natural red light reaching rat RGC mitochondria in situ, subjected to ischemia, proved to be beneficial. A challenge now is to test whether extra red light delivered to the human retina can slow-down RGC loss in glaucoma. Such a methodology has also the advantage of being non-invasive. One very exciting possibility might be in the production of a lens where solar UV light is convertes to add to the amount of natural red light entering the eye.

Blue Light Action on Mitochondria Leads to Cell Death by Necroptosis.

Blue light impinging on the many mitochondria associated with retinal ganglion cells (RGCs) in situ has the potential of eliciting necroptosis through an action on RIP1/RIP3 to stimulate RGC death in diseases like glaucoma and diabetic retinopathy. Cells in culture die when exposed to blue light. The death process is mitochondria-dependent and is known to involve a decrease in the production of ATP, a generation of ROS, the activation of poly-(ADP-ribose) polymerase, the stimulation of apoptosis-inducing factor (AIF) as well as the up-regulation of heme-oxygenase-1 (HO-1). Our present results show that blue light-induced activation of AIF is not directly linked with the stimulation of RIP1/RIP3. Down-regulation of RIP1/RIP3 did not influence AIF. AIF activation therefore appears to enhance the rate of necroptosis by a direct action on DNA breakdown, the end stage of necroptosis. This implies that silencing of AIF mRNA may provide a degree of protection to blue light insult. Also, necrostatin-1 attenuated an increased turnover of HO-1 mRNA caused by blue light to suggest an indirect inhibition of necroptosis, caused by the action of necrostatin-1 on RIP1/RIP3 to reduce oxidative stress. This is supported by the finding that gene silencing of RIP1 and RIP3 has no effect on HO-1. We therefore conclude that inhibitors of RIP kinase might be more specific than necrostatin-1 as a neuroprotective agent to blunt solely necroptosis caused by blue light.

Glaucoma: Focus on mitochondria in relation to pathogenesis and neuroprotection.

Primary open-angle glaucoma (POAG) is a common form of glaucoma in which retinal ganglion cells (RGCs) die at varying intervals. Primary open-angle glaucoma is often associated with an increased intraocular pressure (IOP), which when reduced, can slow down the progression of the disease. However, it is essential to develop better modes of treatments for glaucoma patients. In this overview, we discuss the hypothesis that RGC mitochondria are affected during the initiation of POAG, by becoming gradually weakened, but at different rates because of their specific receptor profiles. With this in mind, we argue that neuroprotection in the context of glaucoma should focus on preserving RGC mitochondrial function and suggest a number of ways by which this can theoretically be achieved. Since POAG is a chronic disease, any neuroprotective treatment strategy must be tolerated over many years. Theoretically, topically applied substances should have the fewest side effects, but it is questionable whether sufficient compounds can reach RGC mitochondria to be effective. Therefore, other delivery procedures that might result in greater concentrations of neuroprotectants reaching RGC mitochondria are being developed. Red-light therapy represents another therapeutic alternative for enhancing RGC mitochondrial functions and has the advantage of being both non-toxic and non-invasive.

Red light of the visual spectrum attenuates cell death in culture and retinal ganglion cell death in situ.

PURPOSE: To ascertain whether red light, known to enhance mitochondrial function, can blunt chemical insults to cell cultures and ischaemic insults to the rat retina. METHODS: Raised intraocular pressure (IOP, 140 mmHg, 60 min) or ischaemia was delivered in complete darkness or in the presence of low intensity red light (16.5 watts/m(2) , 3000 lux, 625-635 nm) to one eye of each rat. Animals were killed at specific times after ischemia and retinas analysis for ganglion cell numbers, the localization of specific antigens or for changes in defined RNAs. RGC-5 cell cultures were also exposed to various chemical insults in the presence or absence of red light. Significant differences were determined by t-test and anova. RESULTS: Elevation of IOP causes changes in the localization of glial fibrillary acid protein (GFAP), calretinin, calbindin, choline acetyltransferase, ganglion cell numbers and an elevation (GFAP, vimentin, HO-1 and mTORC1) or reduction (Thy-1 and Brn3a) of mRNAs in the rat retina. These negative effects to the rat retina caused by ischaemia are reduced by red light. Moreover, chemical insults to cell cultures are blunted by red light. CONCLUSIONS: Low, non-toxic levels of red light focussed on the retina for a short period of time are sufficient to attenuate an insult of raised IOP to the rat retina. Since mitochondrial dysfunctions are thought to play a major role in ganglion cell death in glaucoma, we propose the potential use of red light therapy for the treatment of the disease.

The effect of visual blue light on mitochondrial function associated with retinal ganglions cells.

The retina is the only part of the central nervous system that is exposed to light radiation between 400 and 780 nm. Short wavelength light (SWL) ranging between 400 and 480 nm are absorbed maximally by chromophores located in mitochondria. An abundance of mitochondria are located in retinal ganglion cell (RGC) intraocular axons and photoreceptor inner segments and as a consequence SWL will impact these organelles. The purpose of this article is to summarise the experimental evidence for the possible negative effects of SWL on RGC mitochondria, in situ. The threat of damage to photoreceptor mitochondria may be less than to RGCs, since macular carotenoid, located chiefly in Henle's layer of the photoreceptor inner segment absorbs SWL. The article underlines the hypothesis that SWL contributes to RGC death when these neurones are not in an optimum homoeostatic state as is likely to occur in conditions such as glaucoma and possibly other types of pathologies and even old age. A case therefore exists for the idea that shielding RGCs to slow down visual loss in certain circumstances. This can theoretically be achieved with lenses that reduce transmission of SWL but specifically allow for maximal transmission of 479 nm light to stimulate melanopsin and maintain an optimum sleep/wake cycle.

Targeting mitochondrial dysfunction as in aging and glaucoma.

Neurons depend on their mitochondria for optimum function and become susceptible with age. Mitochondrial function is gradually impaired during aging because more electrons are converted to reactive oxygen species rather than being converted to ATP. Retinal ganglion cell mitochondria are additionally affected in glaucoma because of reduced oxygen delivery. Thus, targeting neuronal mitochondria to enhance their function as in glaucoma and aspects associated with aging provides potential ways of attenuating degenerating diseases. A substance worthy of mention is rapamycin, which affects regulated in development and DNA damage 1 (REDD1), and is known to enhance mitochondrial function. REDD1 appears to be prominent in retinal ganglion cells. An alternative exciting non-invasive approach is to use red light therapy that enhances mitochondrial function.

RTP801 immunoreactivity in retinal ganglion cells and its down-regulation in cultured cells protect them from light and cobalt chloride.

RTP801, a stress-related protein, is activated by adverse environmental conditions and inhibits the activity of mammalian target of rapamycin (mTOR) in promoting oxidative stress-dependent cell death. RTP801 exists both in the mammalian retina and the lens of the eye. Here, we observed RTP801 immunoreactivity in some retinal ganglion cells. Intravitreal injection of cobalt chloride (CoCl2) to mimick hypoxia influenced retinal GFAP (glial fibrillary acidic protein) and heme oxygenase-1 (HO-1) levels, but did not affect RTP801 immunoreactivity or mRNA content relative to GAPDH. However, RTP801 mRNA was elevated when compared with Brn3a mRNA, suggesting that RTP801 is activated in stressed Brn3a retinal ganglion cells. In cultures of RGC-5 cells, RTP801 immunoreactivity was located in the cytoplasm and partly present in the mitochondria. An insult of blue light or CoCl2 increased RTP801 expression, which was accompanied by cell death. However, in cultures where RTP801 mRNA was down-regulated, the negative influence of blue light and CoCl2 was blunted. Rapamycin nullified the CoCl2-induced up-regulation of RTP801 and attenuated cell death. Moreover, rapamycin was non-toxic to RGC-5 cells, even at a high concentration (10µM). The protective effect of rapamycin on RGC-5 cells caused by the inhibition of RTP801 suggests that rapamycin might attenuate retinal ganglion cell death in situ, as in glaucoma.

Maintenance of retinal ganglion cell mitochondrial functions as a neuroprotective strategy in glaucoma.

Loss of vision in glaucoma occurs because retinal ganglion cells (RGCs) die. RGCs have probably more mitochondria than any other neurone in the CNS. It is proposed that stress to mitochondria of individual RGCs is a major trigger of the disease and also provides an explanation why different RGCs die at different times. Pharmacological agents that can maintain mitochondrial functions, in particular to attenuate oxidative stress and to sustain energy production, might therefore provide a novel way of slowing down RGC death and help in the treatment of glaucoma.

Fluorescent labeling of Acanthamoeba assessed in situ from corneal sectioned microscopy.

Acanthamoeba keratitis is a serious pathogenic corneal disease, with challenging diagnosis. Standard diagnostic methods include corneal biopsy (involving cell culture) and in vivo reflection corneal microscopy (in which the visualization of the pathogen is challenged by the presence of multiple reflectance corneal structures). We present a new imaging method based on fluorescence sectioned microscopy for visualization of Acanthamoeba. A fluorescent marker (MT-11-BDP), composed by a fluorescent group (BODIPY) inserted in miltefosine (a therapeutic agent against Acanthamoeba), was developed. A custom-developed fluorescent structured illumination sectioned corneal microscope (excitation wavelength: 488 nm; axial/lateral resolution: 2.6 µm/0.4-0.6 µm) was used to image intact enucleated rabbit eyes, injected with a solution of stained Acanthamoeba in the stroma. Fluorescent sectioned microscopic images of intact enucleated rabbit eyes revealed stained Acanthamoeba trophozoites within the stroma, easily identified by the contrasted fluorescent emission, size and shape. Control experiments show that the fluorescent maker is not internalized by corneal cells, making the developed marker specific to the pathogen. Fluorescent sectioned microscopy shows potential for specific diagnosis of Acanthamoeba keratitis. Corneal confocal microscopy, provided with a fluorescent channel, could be largely improved in specificity and sensitivity in combination with specific fluorescent marking.