M Francesca Cordeiro

Targeting amyloid-β in glaucoma treatment

The development of the devastating neurodegenerative condition, Alzheimers disease, is strongly associated with amyloid-β (Aβ) deposition, neuronal apoptosis, and cell loss. Here, we provide evidence that implicates these same mechanisms in the retinal disease glaucoma, a major cause of irreversible blindness worldwide, previously associated simply with the effects of intraocular pressure. We show that Aβ colocalizes with apoptotic retinal ganglion cells (RGC) in experimental glaucoma and induces significant RGC apoptosis in vivo in a dose- and time-dependent manner. We demonstrate that targeting different components of the Aβ formation and aggregation pathway can effectively reduce glaucomatous RGC apoptosis in vivo, and finally, that combining treatments (triple therapy) is more effective than monotherapy. Our work suggests that targeting the Aβ pathway provides a therapeutic avenue in glaucoma management. Furthermore, our work demonstrates that the combination of agents affecting multiple stages in the Aβ pathway may be the most effective strategy in Aβ-related diseases.

Unexpected low-dose toxicity of the universal solvent DMSO

Dimethyl sulfoxide (DMSO) is an important aprotic solvent that can solubilize a wide variety of otherwise poorly soluble polar and nonpolar molecules. This, coupled with its apparent low toxicity at concentrations <10%, has led to its ubiquitous use and widespread application. Here, we demonstrate that DMSO induces retinal apoptosis in vivo at low concentrations (5 μl intravitreally dosed DMSO in rat from a stock concentration of 1, 2, 4, and 8% v/v). Toxicity was confirmed in vitro in a retinal neuronal cell line, at DMSO concentrations >1% (v/v), using annexin V, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), and AlamarBlue cell viability assays. DMSO concentrations > 10% (v/v) have recently been reported to cause cellular toxicity through plasma membrane pore formation. Here, we show the mechanism by which low concentrations (2–4% DMSO) induce caspase‐3 independent neuronal death that involves apoptosis‐inducing factor (AIF) translocation from mitochondria to the nucleus and poly‐(ADP‐ribose)‐polymerase (PARP) activation. These results highlight safety concerns of using low concentrations of DMSO as a solvent for in vivo administration and in biological assays. We recommend that methods other than DMSO are employed for solubilizing drugs but, where no alternative exists, researchers compute absolute DMSO final concentrations and include an untreated control group in addition to DMSO vehicle control to check for solvent toxicity.—Galvao, J., Davis, B., Tilley, M., Normando, E., Duchen, M. R., Cordeiro, M. F. Unexpected low‐dose toxicity of the universal solvent DMSO. FASEB J. 28, 1317–1330 (2014). www.fasebj.org

Neuroprotection in glaucoma - Is there a future role?

In glaucoma, the major cause of global irreversible blindness, there is an urgent need for treatment modalities that directly target the RGCs. The discovery of an alternative therapeutic approach, independent of IOP reduction, is highly sought after, due to the indirect nature and limited effectiveness of IOP lowering therapy in preventing RGC loss. Several mechanisms have been implicated in initiating the apoptotic cascade in glaucomatous retinopathy and numerous drugs have been shown to be neuroprotective in animal models of glaucoma. These mechanisms and their potential treatment include excitotoxicity, protein misfolding, mitochondrial dysfunction, oxidative stress, inflammation and neurotrophin deprivation. All of these mechanisms ultimately lead to programmed cell death with loss of RGCs. In this article we summarize the mechanisms involved in glaucomatous disease, highlight the rationale for neuroprotection in glaucoma management and review current potential neuroprotective strategies targeting RGCs from the laboratory to the clinic.

Assessment of Rat and Mouse RGC Apoptosis Imaging in Vivo with Different Scanning Laser Ophthalmoscopes

Purpose: We have recently described a novel way of imaging apoptosing retinal ganglion cells in vivo in the rat. This study investigated if this technique could be used in the mouse, and whether the Heidelberg Retina Angiograph II (HRAII) was appropriate. Methods: Retinal ganglion cell (RGC) death was induced by intravitreal injections in rat and mouse eyes using staurosporine. Fluorescent-labeled apoptosing cells were detected by imaging with both the HRAII and a prototype Zeiss confocal scanning laser ophthalmoscope (cSLO). Averaged in vivo images were analyzed and results compared with histologic analysis. Results: Fluorescent points (FPs) used as a measure of RGC apoptosis in vivo were detected in the mouse eye but only with the HRAII and not the Zeiss cSLO. The HRAII was able to detect 62% more FPs in rat than the Zeiss cSLO. Both cSLOs showed peak FP counts at the 5- to 10-μ m range in rat and mouse. Maximal FP counts were detected in the superior and superior temporal regions in the rat, with no obvious pattern of distribution in the mouse. The HRAII was found to have more FP correspondence with histologically identified apoptosing RGCs. Conclusions: To our knowledge, this is the first demonstration of visualized apoptosing RGC in vivo in a mouse. The improved image quality achieved with the HRAII compared with the Zeiss cSLO was validated by histology. This together with its enhanced maneuverability and the fact that it is already commercially available make the HRAII a potential tool for the early detection and diagnosis of glaucomatous disease in patients.

Topical Delivery of Avastin to the Posterior Segment of the Eye In Vivo Using Annexin A5-associated Liposomes

Effective delivery to the retina is presently one of the most challenging areas in drug development in ophthalmology, due to anatomical barriers preventing entry of therapeutic substances. Intraocular injection is presently the only route of administration for large protein therapeutics, including the anti-Vascular Endothelial Growth Factors Lucentis (ranibizumab) and Avastin (bevacizumab). Anti-VEGFs have revolutionised the management of age-related macular degeneration and have increasing indications for use as sight-saving therapies in diabetes and retinal vascular disease. Considerable resources have been allocated to develop non-invasive ocular drug delivery systems. It has been suggested that the anionic phospholipid binding protein annexin A5, may have a role in drug delivery. In the present study we demonstrate, using a combination of in vitro and in vivo assays, that the presence of annexin A5 can significantly enhance uptake and transcytosis of liposomal drug carrier systems across corneal epithelial barriers. This system is employed to deliver physiologically significant concentrations of Avastin to the posterior of the rat eye (127 ng/g) and rabbit retina (18 ng/g) after topical application. Our observations provide evidence to suggest annexin A5 mediated endocytosis can enhance the delivery of associated lipidic drug delivery vehicles across biological barriers, which may have therapeutic implications.

Real-Time In Vivo Imaging of Retinal Cell Apoptosis after Laser Exposure

PURPOSE To investigate whether the detection of apoptosing retinal cells (DARC) could detect cells undergoing apoptosis in a laser model of retinal damage. METHODS Laser lesions were placed, with the use of a frequency-doubled Nd:YAG laser, on the retina in 34 eyes of anesthetized Dark Agouti rats. Lesion size and laser-induced retinal elevation were analyzed using in vivo reflectance imaging. Development of retinal cell apoptosis was assessed using intravitreal fluorescence-labeled annexin 5 in vivo with DARC technology from baseline until 90 minutes after laser application. Histologic analysis of retinal flat mounts and cross-sections was performed. RESULTS The lateral and anteroposterior depth extension of the zone of laser damage was significantly larger for higher exposure settings. A strong diffuse signal, concentrated at the outer retina, was seen with DARC for low exposures (<300 ms and <300 mW). In comparison, higher exposures (>300 ms and >300 mW) resulted in detectable hyperfluorescent spots, mainly at the level of the inner retinal layers. Dose-dependent effects on spot density and positive correlation of spot density between lesion size (P < 0.0001) and retinal elevation (P < 0.0001) were demonstrated. Histology confirmed the presence of apoptosing retinal cells in the inner nuclear and the ganglion cell layers. CONCLUSIONS This is the first time that DARC has been used to determine apoptotic effects in the inner nuclear layer. The ability to monitor changes spatially and temporally in vivo promises to be a major advance in the real-time assessment of retinal diseases and treatment effects.

Assessment of neuroprotection in the retina with DARC.

Currently, assessment of new drug efficacy in glaucoma relies on conventional perimetry to monitor visual field changes. However, visual field defects cannot be detected until 20–40% of retinal ganglion cells (RGCs), the key cells implicated in the development of irreversible blindness in glaucoma, have been lost. We have recently developed a new, noninvasive real-time imaging technology, which is named DARC (detection of apoptosing retinal cells), to visualize single RGC undergoing apoptosis, the earliest sign of glaucoma. Utilizing fluorescently labeled annexin 5 and confocal laser scanning ophthalmoscopy, DARC enables evaluation of treatment effectiveness by monitoring RGC apoptosis in the same living eye over time. Using DARC, we have assessed different neuroprotective therapies in glaucoma-related animal models and demonstrated DARC to be a useful tool in screening neuroprotective strategies. DARC will potentially provide a meaningful clinical end point that is based on the direct assessment of the RGC death process, not only being useful in assessing treatment efficacy, but also leading to the early identification of patients with glaucoma. Clinical trials of DARC in glaucoma patients are due to start in 2008.

Current perspective of neuroprotection and glaucoma.

Glaucoma is the second leading cause of blindness worldwide and is most notably characterized by progressive optic nerve atrophy and advancing loss of retinal ganglion cells (RGCs). The main concomitant factor is the elevated intraocular pressure (IOP). Existing treatments are focused generally on lowering IOP. However, both RGC loss and optic nerve atrophy can independently occur with IOP at normal levels. In recent years, there has been substantial progress in the development of neuroprotective therapies for glaucoma in order to restore vital visual function. The present review intends to offer a brief insight into conventional glaucoma treatments and discuss exciting current developments of mostly preclinical data in novel neuroprotective strategies for glaucoma that include recent advances in noninvasive diagnostics going beyond IOP maintenance for an enhanced global view. Such strategies now target RGC loss and optic nerve damage, opening a critical therapeutic window for preventative monitoring and treatment.

Clinical Evidence for Neuroprotection in Glaucoma

Glaucoma is treated by lowering intraocular pressure (IOP). However, numerous laboratory studies have shown that experimental glaucoma can be helped by therapies that act by mechanisms other than lowering IOP. Such therapies are termed “neuroprotective” because the targets are the neurons affected in glaucomatous optic neuropathy. Until recently there has been no level I evidence that any neuroprotective therapy is effective in patients with glaucoma.1 With the recent online publication of the results of the Low Pressure Glaucoma Treatment Study2 (LoGTS), there are now data that the α2 agonist brimonidine, a drug previously shown to be neuroprotective in the laboratory, may also have a beneficial effect on visual function independent of IOP lowering. This commentary analyzes the implications of LoGTS for our understanding of neuroprotection. LoGTS was a randomized double-masked, multicenter study comparing visual field progression in normal-tension glaucoma patients treated with brimonidine or timolol. Subjects had visual field and optic nerve head evidence of glaucomatous optic neuropathy and a diurnal IOP < 21 mm Hg. Randomization was in a 4:3 (brimonidine:timolol) ratio to account for an expected increased dropout in the brimonidine group from ocular allergy. The primary outcome measure was visual field progression at 3 or more points by pointwise linear regression and confirmed on 3 successive fields. Sample size was based on an 80% power to detect a 25% difference in visual field progression at α=0.05. Follow-up was up to 4 years with visual fields every 4 months. Mean (±SEM) follow-up was 30±2 months. Of the 178 analyzed patients, 9.1% of the brimonidine group and 39.2% of the timolol group progressed (p=0.001). Comparable visual field progression results were seen when analyzed by glaucoma change probability mapping or the 3-omitting method for pointwise linear regression. IOP decrease was similar in the two groups, implying that the difference in progression might be unrelated to effects on IOP. This impressive difference in visual field progression in two therapies with similar IOP lowering profiles is tempered by several issues. First, the higher incidence of ocular allergy in the brimonidine group (20%) led to significant drop-out in the first year, and could have masked a subgroup of progressing patients that were not subsequently analyzed. If so, this would imply that visual field progression and incidence of ocular allergy are highly associated, which is uncommonly observed. Nonetheless, the apparent neuroprotective effects of brimonidine may only be applicable to patients who do not develop ocular allergy. Ocular allergy insufficient to cause a subject to drop-out could unmask group assignment to the treating ophthalmologist, although this is less concerning because the visual field analysis was masked. Second, the diurnal effects of brimonidine and timolol could have differed, with less nocturnal IOP reduction with timolol compared to brimonidine. Against this are studies demonstrating almost identical IOP lowering over 24 hours with the two drugs3 and insignificant nocturnal IOP lowering with brimonidine.4 Ocular perfusion pressure (diastolic blood pressure minus IOP) in the first study was even lower with brimonidine than timolol, implying that changes in ocular perfusion were not responsible for the apparent neuroprotective effects observed in LoGTS. Third, in retrospect, subjects in both groups were undertreated. The proportion with a ≥ 20% decrease in IOP at the time of progression was only 44% in the brimonidine group and 39% in the timolol group. In comparison, target reduction of IOP was 30% in the Collaborative Normal Tension Glaucoma Study.5 The baseline diurnal IOP in progressing brimonidine subjects was higher than timolol subjects (16.9±2.4, n=9 vs. 15.4±2.5, n=31), indicating that there was still a role for further IOP lowering in addition to neuroprotection. Additional IOP lowering would likely have made it more difficult to detect a neuroprotective effect due to brimonidine. Fourth, the perceived lower progression rate in brimonidine-treated subjects could be the result of a paradoxically higher progression rate in timolol-treated subjects. Timolol may have effects on neural or vascular physiology that theoretically could be deleterious to the optic nerve. On the other hand, there is no reason to believe that timolol is actually harmful in patients with glaucoma, even though it could explain the results obtained. This could be explored further by comparing progression rates in CNTGS between timolol-treated normal-tension glaucoma subjects and untreated subjects. The apparent neuroprotective effect observed in the LoGTS trial should be placed in the context of clinical trials that did not demonstrate neuroprotective efficacy, including those in other neurologic diseases.6,7 These trials were preceded by laboratory studies demonstrating potent neuroprotective effects in animals, yet translation to the clinic did not occur. Some reasons for this “Loss in Translation” are discussed elsewhere.8,9 The results of the LoGTS are encouraging, and one interpretation is that the drug studied was associated with a neuroprotective effect. Why this result differed from other studies of neuroprotection is unknown, and could reflect study design, the disease studied, the drug used, our inability to measure neuroprotection directly, or a combination thereof. However, confirmation of the neuroprotection paradigm by other randomized clinical trials is necessary to increase confidence that this and other non-IOP lowering approaches are viable therapies for glaucoma and other optic neuropathies.

Preservative-free treatment in glaucoma: who, when, and why?

Purpose To review and summarize the available literature on the effect of preservatives on the eye, to provide practical guidance for the clinical assessment of the ocular surface in glaucoma patients, and to define patient populations that might benefit from preservative-free topical intraocular pressure (IOP)–lowering agents. Methods This manuscript is based on a combination of a literature review on preservatives and the eye and expert opinion from glaucoma specialists with an interest in ocular surface disease. Results There is an increasingly recognized association between eyedrop preservatives and ocular surface disease. Preservative-free therapy is now available for a wide range of active compounds, although there are still some misconceptions regarding their appropriate use. For patients treated topically for glaucoma or ocular hypertension, a rough estimate could be that 20% may need treatment with topical IOP-reducing agents that are free from preservatives. Conclusions This review provides an up-to-date account of the literature regarding preservatives and the eye, as well as suggestions and recommendations on to when to use preservative-free antiglaucoma treatment.