Real-Time Imaging of Retinal Cell Apoptosis by Confocal Scanning Laser Ophthalmoscopy and Its Role in Glaucoma

Glaucoma is one of the leading causes of irreversible blindness in the world. It is characterized by the progressive loss of retinal ganglion cells (RGCs), mainly through the process of apoptosis. Glaucoma patients often come to clinical attention when irreversible loss of visual function has been already established; therefore, early recognition of RGC apoptosis is inordinately important in disease prevention. The novel technology called Detection of Apoptosing Retinal Cells (DARC) allows real-time in vivo quantification of apoptosing cells through the use of a fluorescent biomarker and a confocal scanning ophthalmoscope. A recent Phase I clinical trial has evaluated the safety of DARC and its ability to detect retinal apoptosis in glaucoma patients and healthy volunteers. Results suggest that DARC may have potential in the early detection of glaucoma, which could help alleviate the medical, social, and economic burden associated with this blinding condition.

Glaucoma is one of the leading causes of irreversible blindness in the world. It is characte rized by the progressive loss of retinal ganglion cells (RGCs), mainly through the process of apoptosis. Glaucoma patients often come to clinical attention when irreversible loss of visual function has been already established; therefore, early recognition of RGC apoptosis is inordinately important in disease prevention. The novel technology called Detection of Apoptosing Retinal Cells (DARC) allows realtime in vivo quantification of apoptosing cells through the use of a fluorescent biomarker and a confocal scanning ophthalmoscope. A recent Phase I clinical trial has evaluated the safety of DARC and its ability to detect retinal apoptosis in glaucoma patients and healthy volunteers. Results suggest that DARC may have potential in the early detection of glaucoma, which could help alleviate the medical, social, and economic burden associated with this blinding condition.
Keywords: glaucoma, annexin, retinal ganglion cells, imaging, apoptosis inTRODUCTiOn Glaucoma is one of the leading causes of blindness with over 70 million people affected worldwide and projected to be 80 million by 2020 (1). It is as an optic neuropathy characterized by cupping of the optic disk, thinning of the retinal nerve fiber layer, and visual field loss (2), caused by the death of retinal ganglion cells (RGCs) (3) predominantly mediated by the mechanism of programmed cell death, otherwise known as apoptosis (4). During apoptosis, cascades of enzymes are activated leading to a series of biochemical and morphological changes, including cell and nuclear shrinkage, chromatin condensation, DNA fragmentation, and bleb formation (5). It is at the level of the cell membrane that one of the key mechanisms of apoptosis takes place: the translocation of phosphati dylserine (PS) from the inner to the outer leaflet of the cell membrane (6). Although exposed PS acts as an "eat me" signal to phagocytes, it is also where the endogenous molecule Annexin 5 (ANX) binds (7). This property has been exploited for many years in the detection of apoptosis using fluorescently labeled ANX and recently utilized in the development of a novel in vivo retinal imaging techno logy called Detection of Apoptosing Retinal Cells (DARC) technology (8)(9)(10)(11). DARC has potential in the early identification of patients with glaucoma before irreversible damage occurs (8). This review summarizes the development of the DARC technology and discusses its role in glaucoma and neurodegeneration.
FiGURe 1 | Visualization of nerve cell apoptosis. Retinal ganglion cell (RGC) apoptosis could be detected in vivo by using a modified cLSO with an argon laser (488 nm) for illumination and a wide bandpass filter with short wavelength cutoff of a 521nm filter and intravitreal AlexaFluor 488 fluorescentlabeled annexin 5. This was a model of staurosporine (SSP) induced apoptosis. This rat's eye was treated with intravitreal SSP (0.5 µg). This retinal image shows extensive RGC apoptosis (white spots, annexin 5positive apoptotic cells).

BiOMARKeRS AnD APOPTOSiS
The annexins are a family of Ca 2+ dependent proteins which bind to anionic phospholipids (12). Annexin V is a protein with a high affinity for PS (13). Translocation of PS from the inner to the outer leaflet of the cellular membrane is an early event in apoptosis and its identification is a potential biomarker for early detection of cell death. ANX has been used for many years in diagnostic in vitro assays for the identification of apoptotic cells in laboratory samples. In the last two decades, ANX has been conjugated to radioactive materials, such as Technetium99, and used clinically to detect apoptosis in many conditions including breast cancer (14), nonHodgkin's lymphoma (15), myocardial infarction (16), and rectal cancer (17).

THe DeveLOPMenT OF DARC
The technique known as DARC has been developed over the last few years to initially to assess the degree of RGC apoptosis in experimental models of glaucoma (11,18). More recently, it has also been used to investigate the occurrence of retinal cell apoptosis in other neurodegenerative disease models (19)(20)(21).
Although the loss of RGCs underlies the pathogenesis of glaucoma and other optic neuropathies, its degeneration is known to occur many years before any visual field defects are identified (22,23). Patients are typically managed by serial measurements of intraocular pressure (IOP), retinal nerve fiber layer and optic disk imaging, as well as with visual field tests. However, these parameters alone may not be sufficient for monitoring glaucoma progression, as progressive visual field loss has been demonstrated in patients despite "wellcontrolled" IOP (24,25). The gold standard for detecting glaucomatous changes remains standard automated whiteonwhite perim etry. It is a test that requires good patient cooperation, is time consuming, and can be inconsistent and variably interpreted (26). Visual field changes may not become apparent until the disease is well established and by then, irreversible damage has occurred.
The first in vivo use of DARC technology was demonstrated in experimental glaucoma in 2004 (20) where realtime visualiza tion of a single nerve cell apoptosis through the eye's clear optical media was recorded (Figure 1). Validation of the technique was established using ocular hypertensive and optic nerve transec tion models (20). Histological analyses were used to confirm the in vivo findings. Repeated assessment of the same eye over time with DARC has also shown promising results for its application in evaluating potential neuroprotective strategies (27,28).
Detection of apoptosing retinal cell has been validated in a few other experimental models of neurodegenerative disease and optic neuropathy, where is has been used to assess disease severity and treatment efficacy. Some studies investigate different neuroprotective strategies in a rat model of glaucoma (28) as well as novel potential methods of reversing optic neuropathy (29) as summarized in the table below ( Table 1).
Over the years, the technology has evolved. To date, the DARC technique is composed of intravenous injection of an infrared fluorescently labeled ANX (ANX776), followed by retinal imaging using specific wavelengths (excitation: 786 nm; photodetector barrier filter: 800 nm; emission: 815 nm) using a commercially available confocal scanning laser ophthalmoscope (cSLO) and indocyanine green angiography settings. One of the key elements of the confocal imaging instrumentation is the ability to focus on the inner retina layers, which permits capturing of the signal coming from the RNFL and GCL layers (33)(34)(35).
Aside from DARC, other methods of imaging and monitor ing cellular apoptosis have been studied. For example, TCapQ is an experimental technique which utilizes an intracellular nearinfrared fluorescent peptide probe for the in vivo visualiza tion of apoptotic RGCs. It involves using a cellpenetrating Tat peptide conjugated to an effector caspase recognition sequence, a quencher and a fluorophore (AlexaFluor 488) that is activated by caspase 3 and 7 in the cytoplasm of apoptotic cells (36). This technology detects effector caspase activity, which signifies an apoptotic cascade activation (37). However, this method is complex and requires intracellular delivery of molecular imaging probes to enable detection of apoptosis. It also involves intravit real administration, which is invasive and not the most suitable method for clinical translation.
AAV vectors are a method of targeting gene expression, which can be used for fluorescently labeling RGCs and studying RGC number in vivo. These vectors have the ability to incorporate different promoters, which allows for high specificity in targeting RGCs. AAV vectors are administered via intravitreal injections, with the aim of quantifying cell density in vivo (38). Again, intra vitreal injections are invasive not the best option for monitoring patients. Also, the use of viral vectors and gene delivery is still associated with complications, which would make this an unsuit able test to use routinely in patients. Adaptive optics scanning light ophthalmoscopy (AOSLO) is a method of imaging the RGC layer in the human retina without using any cell labeling techniques. It is possible to use this imag ing method to study the RGC layer in glaucoma using its ability to visualize individual cells, although RGC imaging is difficult due to their low contrast edges. Subcellular resolution can also demonstrate the changes that subcellular structures undergo during apoptosis in glaucoma (39). However, AO imaging requires heavy image processing, and optimal images are very sensitive to focus position and difficult to obtain. Moreover, it is impossible to image the whole retina using AOSLO, as the field of view is very limited. This again makes it a suboptimal method for monitoring overall glaucoma progression.
Detection of apoptosing retinal cell allows for a less invasive, patientacceptable method of imaging RGCs in a wide field of view in the retina, which could be clinically useful in monitoring progression in patients with optic neuropathy.

CLiniCAL USe OF DARC
Early detection of any disease is paramount and is one of the most important aspects of any diagnostic test (40). In glaucoma, where functional visual field damage is only detectable after at least 40% of RGCs are lost, there is an unmet need for an early diagnostic tool (41). Apoptosis is a key process in glaucoma; therefore, its evaluation should be of utmost importance. Prior to DARC techno logy, the only method of assessing apoptosis definitively was through post mortem histology (22,33). Experimental data from animal glaucoma models have shown the potential for DARC to predict the rate of disease progression in humans (20,33,34). DARC could provide a rapid and effective means of detecting apoptotic RGCs that would allow earlier detection and thus, acting as a biomarker for novel treatment strategies.
Clinical trials of DARC have involved the intravenous (IV) administration of ANX776 and an assessment of retinal cell apoptosis with commercially available cSLO devices. IV admin istration of angiographic contrast solutions, such as sodium fluorescein and indocyanine green, is a wellestablished method in ophthalmology for imaging retinal diseases (42). Use of IV radiolabeled Annexin V has been shown to be safe in multiple clinical trials and indications, including heart transplantation (43), breast cancer (14), nonHodgkin's lymphoma (14), and myocar dial infarction (15).
A recent Phase I study assessed the use of IV ANX776 to identify retinal cell apoptosis in humans (8). Briefly, the study has shown that apoptosing retinal cells can be seen in human patients as white fluorescent spots against a dark retinal background. The total number of spots makes up the DARC Count which gives a precise quantitative evaluation of cell damage at a given time point (Figure 2). In this Phase I trial, half the enrolled subjects were eight healthy volunteers and the other half were eight patients with progressive glaucoma. Although the trial was designed pri marily to assess the safety and tolerability of ANX776 in patients, it showed that the DARC Count was significantly increased in glaucoma patients compared to healthy volunteers. Furthermore, the DARC Count correlated with increasing rates of glaucoma tous progression. Importantly, ANX776 was shown to be safe and well tolerated with no serious adverse effects, such as anaphylaxis, arrhythmias, loss of consciousness, etc. There were single cases of mild and selflimiting adverse effects, for example, hematoma at the cannulation site and discomfort during phlebotomy, which were likely unrelated to ANX776. ANX776 is rapidly cleared from the human body and has a short halflife of about 10-36 min. The optimal dosing was determined based on the dose the better cor related with the highest level of DARC Counts (0.4 mg). Overall, the trial showed the potential of DARC as a method for detecting and assessing the level of neurodegeneration in glaucoma in vivo using RGC apoptosis as an indicator.
Currently, the DARC Phase II trial is being analyzed, having completed the patient intervention stage. It primarily aims to evaluate the efficacy of DARC in visualizing apoptotic retinal cells in patients with neurodegenerative diseases, including glaucoma, agerelated macular degeneration, optic neuritis, and Down's syndrome. Increasing evidence suggests the eye is involved in CNS diseases, such as Alzheimer's disease (AD) and Parkinson's diseases (PD). In particular it has been demonstrated, experimentally, that DARC can detect retinal cell death in vivo in a mouse model of AD (3XTGAD) (19). Furthermore, DARC has been used to assess RGC apoptosis in an experimental model of PD, showing that retinal changes can be identified before classical histological changes in the brain occur (21).

FUTURe DiReCTiOnS
Detection of apoptosing retinal cell is currently being evaluated in clinical trials, and its validation will become increasingly important as it compared to existing gold standards in identifying and monitoring disease progression.
Although not yet assessed clinically, experimentally DARC has been shown useful in test ing treatment efficacy in a variety of different neurodegenerative conditions such as AD (44) and PD (19,21,28). In PD, it was shown that retinal changes may be a good surrogate biomarker of disease progression and treatment efficacy (21). Similarly, DARC technology could potentially be applied to studying rare but visually devastating mitochondrial optic neuropathies such as Leber's hereditary optic neuropathy and dominant optic neu ropathy (45). In the next few years, it is anticipated that DARC will be investigated in clinical treatment trials with an exploratory endpoint.
As DARC continues to develop, its application to glaucoma as well as other optic neuropathies and neurodegenerative disease represents a novel in vivo approach to assessing disease progression. Earlier detection of glaucoma could potentially allow for more efficacious treatment, and reduce the global health burden of this leading cause of blindness.

AUTHOR COnTRiBUTiOnS
EY and TAM drafted the article. EY, EN, and MC edited the article for submission.