The Future of Imaging in Detecting Progression

Fabio Lavinsky, MD, MBA, Gadi Wollstein, MD, Jenna Tauber, BS, Joel S. Schuman, MD

Ocular imaging has been heavily incorporated into glaucoma management and provides important information that aids in the detection of disease progression. Longitudinal studies have shown that the circumpapillary retinal nerve fiber layer is an important parameter for glaucoma progression detection, whereas other studies have demonstrated that macular parameters, such as the ganglion cell and optic nerve head pa- rameters, also are useful for progression detection. The introduction of novel technologies with faster scan speeds, wider scanning fields, higher resolution, and improved tissue penetration has enabled the precise quantification of additional key ocular structures, such as the individual retinal layers, optic nerve head, , and lamina cribrosa. Furthermore, extracting functional information from scans such as blood flow rate and oxygen consumption pro- vides new perspectives on the disease and its progression. These novel methods promise improved detection of glaucoma progression and better insight into the mechanisms of progression that will lead to better targeted treatment options to prevent visual damage and blindness. Ophthalmology 2017;124:S76-S82 ª 2017 by the American Academy of Ophthalmology

Glaucoma is a multifactorial optic neuropathy characterized Despite the usefulness of OCT, there are also challenges in by structural damage of retinal ganglion cells (RGCs) and glaucoma detection and its progression with this technology. their axons that is associated with vision loss and may lead These challenges are the result of structural variability in to irreversible blindness.1 Because glaucomatous damage is healthy , overlap in structural measurements between irreversible and effective treatment is available to halt healthy and early glaucomatous eyes, and abnormal- further damage, glaucoma management should be appearing eyes that do not show any evidence of disease optimized with precise micrometer-scale quantifications of progression over time (e.g., physiologic cupping). Addition- ocular structures that improve detection of the disease and ally, normal age-related structural loss can confound the its progression.2e4 The introduction of OCT technology interpretation of longitudinal glaucoma assessment.12e15 more than 20 years ago provided in vivo detailed visuali- OCT comparison with functional visual testing, such as zation of the optic nerve head (ONH) and and enabled standard automated perimetry (SAP), also introduces a sub- the quantitative evaluation of these structures.5,6 Circum- stantial challenge when assessing disease progression. The papillary retinal nerve fiber layer (RNFL) thickness is a measurement variability of SAP is high and can be influenced common OCT measurement that provides comprehensive by many confounding factors.16,17 Additionally, the associ- evaluation of all RGCs in an as they converge into the ation between structural and functional abnormalities in ONH.2 When measured with spectral-domain OCT, the glaucoma varies with disease severity, which also should be RNFL has been shown to differentiate between healthy and considered when assessing progression with these methods. glaucomatous eyes.7 The steady evolution of OCT In early stages of the disease, the high variability in SAP technology has led to imaging with better resolution, measurements often delays the possibility of detecting func- higher scanning speeds, and advanced imaging patterns tional progression at a time when changes may be noted with that has improved the reliability of OCT measurements structural assessment. A so-called tipping point was reported, and allowed for detection of minute changes that can illustrating the point in disease severity before which struc- improve the sensitivity of progression detection. tural and functional changes often disagree and after which Assessment of glaucoma progression usually is based on there is a strong association between the two.18 Other studies event or trend analysis. Event-based progression determines have demonstrated that the SAP 24-2 testing strategy misses when a measurement exceeds a pre-established threshold for more central points compared with the 10-2 strategy in early change from baseline. Trend-based analysis quantifies the glaucomatous losses.19 Given the improved ability to detect rate of a parameter’s progression over time.2,8 Conven- macular thinning of RNFL and in recent tionally, structural progression has been assessed using iterations of the technology, an improved agreement RNFL measurements; however, longitudinal studies have between macular structure and visual function may be shown that other parameters, including the macular ganglion present in earlier stages than previously described. cell complex, ganglion cell-inner plexiform layer, and ONH parameters (rim area, cup area, and cup-to-disc ratio), also 9e11 are useful for evaluating glaucoma progression. Statement of Potential Conflict of Interest and Funding/Support: See page S82.

S76 ª 2017 by the American Academy of Ophthalmology https://doi.org/10.1016/j.ophtha.2017.10.011 Published by Elsevier Inc. ISSN 0161-6420/17 Lavinsky et al The Future of Imaging

In advanced disease, the opposite situation occurs in resulting from the movement of red blood cells.31 OCT which assessment of the RNFL, as measured with OCT, is angiography allows the peripapillary, ONH, and macular less sensitive than SAP in detecting progression after a vasculature to be evaluated at various depths, exposing e minimum measurable thickness is reached; this is referred to several vascular networks.32 34 The peripapillary and as the floor effect.20,21 It should be noted, however, that ONH capillaries have been demonstrated to be associated there are also limitations in visual field diagnosis of pro- with glaucoma.35e37 The flow index (mean decorrelation gression when mean sensitivities are between 19 and 15 dB value on the en face retinogram) and vessel density (area and below because of a reduction in the asymptotic occupied by large vessels and microvasculature) have been maximum response.22,23 Furthermore, longitudinal analysis shown to be associated with disease severity as reflected by of OCT macular and ONH parameters have demonstrated SAP global indices.38 Additional studies demonstrated a their ability to detect structural progression even at stages reduction in peripapillary vessel density in the where the peripapillary RNFL reached the floor effect hemispheres corresponding to hemifield defects in visual thickness.24 In a cohort of advanced glaucoma, defined by fields.39,40 In eyes with glaucoma and a single-hemifield visual field mean deviation of -21 dB, another study defect, diminished vascular density of the macular and showed a significant rate of ganglion cell-inner plexiform peripapillary regions also was demonstrated in unaffected layer thinning in 31% of the eyes.25 It also has been hemifields.41 OCT angiography of eyes with glaucoma and demonstrated in a group of patients with advanced LC defects portrayed diminished vascular density glaucoma that the ganglion cell-inner plexiform layer had corresponding to the locations of these defects.42 a rate of change of -0.21 mm/year and that the dynamic A recent longitudinal study with a short follow-up period range above the measurable floor was larger than the of less than 14 months showed a significantly faster rate of peripapillary RNFL and minimum rim width.26 Therefore, change of the superficial capillary plexus density in glau- macular OCT measurements provide an alternative for comatous eyes compared with glaucoma suspects and objective and quantitative monitoring of patients with healthy eyes.43 This promising initial result should be advanced glaucoma. corroborated by additional studies evaluating changes in To address these challenges and to explore opportunities other regions. in glaucoma progression detection, several new technolo- gies, methods, and image processing tools are being studied. Doppler OCT The assessment of the structure-function association with Color Doppler imaging shows significantly lower blood newer software and the introduction of advanced statistical flow velocities and higher resistive indexes in the central methods to analyze and predict progression from large retinal artery, short posterior ciliary arteries, and ophthalmic longitudinal data sets are promising tools to be incorporated artery in patients with clinically worsening primary open- into clinical practice. Additionally, novel methods and angle glaucoma (POAG).44,45 Doppler OCT previously technologies are enabling the introduction of new bio- was reported to measure total flow around the ONH, but markers that may enhance the assessment of glaucoma small vessel flow could not be detected.46 Recently, high- progression further and may improve the understanding of speed en face Doppler OCT showed reduced total retinal the disease pathophysiologic features. blood flow in diabetic eyes compared with healthy eyes.47 The value of vascular flow quantification in assessing Novel OCT Technologies and Applications glaucoma progression is yet to be determined.

Swept-Source OCT Adaptive Optics Swept-source OCT is a newer generation of OCT that uses a The incorporation of adaptive optics (AO) systems into ophthalmic imaging has improved achievable image quality short-cavity swept laser with a tunable wavelength of oper- 48 ation. It has a longer central wavelength (1050 nm) further. AO corrects for the monochromatic optical compared with conventional spectral-domain OCT (840 nm), aberrations of the eye, thus improving the resolution of ocular imaging technologies.49 For example, AO OCT providing deeper penetration and eliminating the depth- m dependent signal drop-off observed with earlier generations improves transverse resolution from the typical 20 mto approximately 5 mm.50 Pruning of RGC dendrites has been of OCT. These capabilities enable imaging of deeper ocular 51 structures, such as the choroid and lamina cribrosa shown to be an early indicator of glaucomatous damage ; (LC).27e29 Additionally, improved visualization of the RGC using an AO system conjugated with scanning laser ophthalmoscopy, investigators showed that imaging of layer allowed for the introduction of a model to estimate 52 RGC quantity, which may be useful for understanding the individual RGC bodies is possible in humans. Additional longitudinal structure-function association.30 studies with AO systems have demonstrated changes in the LC microstructure in glaucomatous eyes.53,54 Others have fi OCT Angiography allowed detection of damage down to individual retinal ber layer bundles and the inner and outer retinal layers in glau- OCT angiography uses decorrelation motion contrast be- coma.55,56 However, this technology still presents some bar- tween rapidly repeated OCT cross-section scans to docu- riers to its widespread adoption, namely high cost, limited ment retinal vessels. This is achieved by detecting variations field of view, narrow depth of focus, and extended acquisition in the intensity, phase properties, or both of the OCT signal time.48

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Other Experimental OCT Systems 850 nm. By combining images along a range of wavelengths, maps can be generated to reflect the oxygenated and deoxygenated blood Other experimental OCT systems that may be useful for content within the vasculature.68 Retinal oxygen saturation was glaucoma follow-up are polarization-sensitive OCT and reported to be correlated positively with visual field and RNFL visible light OCT. Polarization-sensitive OCT generates im- thickness; a negative correlation was seen between oxygen ages with tissue-specific contrast based on properties that alter saturation in the choroid plexus and RNFL thickness.69 the polarization state, such as birefringence (, RNFL), Multispectral imaging also has been used to measure increased venular retinal oxygen saturation in POAG compared with polarization preservation (photoreceptors), and depolarization 70 (retinal pigment epithelium).57 Visible light OCT can provide healthy participants. Measurements using multispectral imaging have illustrated differential light absorption in neuroretinal rim both structural and functional information from the same tissue that is associated with visual field sensitivity in POAG.71 scan. The device uses a broad wavelength within the visible light spectrum that can be parsed to determine oxygenated Magnetic Resonance Imaging and deoxygenated blood, and thus to determine the oxygen Because glaucoma is an optic neuropathy often suggested to have a consumption of the tissue as a functional surrogate. The 72 usefulness of these novel biomarkers for longitudinal central nervous system component, magnetic resonance imaging assessment is yet to be determined. (MRI) technology has been introduced as a potential tool for fi glaucoma progression detection. Diffusion tensor imaging MRI Conventional RNFL assessment is based on prede ned studies have demonstrated abnormalities in the optic radiation sectoral analysis; however, in some instances, this approach and optic nerve that correlate with glaucoma severity.73,74 may overlook localized defects and their progression. In vivo MRI on glaucomatous animal models has been used suc- Focusing on the affected area, also known as the region of cessfully to demonstrate increased retinal and choroidal blood flow interest, was tested as an alternative approach in eyes with with topical dorzolamide therapy.75 Each of these findings supports disc hemorrhage, a high-risk population for progression.58 the role of MRI as a potential method in detecting glaucoma The region of interest approach was superior to global progression. RNFL thickness in detection of localized loss. This approach also could be applied for measuring and Magnetization Transfer Imaging monitoring disease progression in other structures. Magnetization transfer imaging is an MRI technique that detects The improvements to OCT scan speed and resolution macromolecules and their associated water molecules. In POAG have resulted in better imaging of the ONH. Parameters patients, the ability to detect axonal and myelin loss has been used such as rim area, vertical rim thickness, and vertical cup-to- to reveal optic nerve atrophy and degeneration of the ratio have been shown to allow detection of glaucoma pathway.76 Researchers have used this tool in humans to identify progression.59 In cross-sectional studies, minimum rim increased demyelination in the optic chiasm, calcarine fissure, geniculocalcarine, and striate area in POAG eyes compared with width showed a high association with glaucomatous func- 76,77 tional changes on SAP.60,61 control eyes. Although this is a step toward understanding the pathogenesis of optic nerve changes in glaucoma, the appli- The LC has been suggested as an important target for cability of magnetization transfer imaging toward detecting POAG axonal injury in glaucoma because certain structural and progression has not been delineated. biomechanical features within this structure may lead to neuronal damage. Advanced imaging offered by current Functional Magnetic Resonance Imaging technologies allows for in vivo evaluation of LC structures, including prelaminar tissue, LC thickness, localized LC Functional MRI is capable of producing cortical mapping of visual function.78 studies have revealed the ability of defects, LC insertion depth, and posterior displace- 53,54,62,63 functional MRI to detect inner retinal layer thinning, optic nerve ment. Swept-source OCT was used for a cupping, and reduced visual cortex activity before any evidence 3-dimensional evaluation of the LC in normal-tension of visual field impairment on SAP testing.79 glaucoma that demonstrated a significant correlation be- tween LC thickness and both RNFL thickness and the mean Proton Magnetic Resonance Spectroscopy deviation of the visual field.64 Statistically significant differences in LC microstructure have been reported Pursuing additional functional imaging techniques, researchers also 65 have used proton magnetic resonance spectroscopy to assess cross- between healthy and glaucomatous eyes. sectional glaucoma diagnosis. These methods have revealed a The biomechanics of the LC determine tissue deforma- correlation between choline metabolism disruptions within the vi- tion in response to its pressure environment. Locations with sual field and disease severity on diffusion tensor MRI and visual marked deformations may strangulate the passing axons, field assessment.79 Longitudinal studies with larger sample sizes leading to impaired functionality.66,67 Given the LC’s are still needed before conclusions can be drawn about the pivotal role in glaucoma pathophysiologic characteristics, its clinical usefulness of these methods. parameters are potential biomarkers for glaucoma progres- sion management. Combining Structure and Function

Other Ophthalmic Imaging Methods As discussed previously, the association between structural and functional changes varies along the spectrum of glau- Multispectral Imaging coma severity. Novel technologies now offer automated and Multispectral imaging is a noninvasive tool for examining layers of precise colocalization of structural and predicted functional the retina and choroid by using light wavelengths from 550 to information as a first step toward better determination of

S78 Lavinsky et al The Future of Imaging disease progression.80,81 Another approach is using struc- continue to assess the potential role of detection of tural and functional information to estimate the number of apoptosing retinal cells in evaluating disease progression. RGC axons in the eye. This model estimates the structural RGC axon quantity from OCT RNFL thickness. Functional Preclinical Biomarkers estimates of RGCs are based on conversion models devel- oped from histologic studies of monkeys.82e84 Because Fluorescently tagged cholera toxin subunit B, injected SAP is less sensitive in detecting early glaucomatous pro- intravitreously, was used in mice to label RGCs success- gression, the combined technique uses a weighted scale that fully.97 Although this technique has been deemed safe in takes into account the differences in OCT and SAP per- humans,98 its use has been limited thus far by its low formances in different stages of disease. This method was specificity. In mouse models many displaced amacrine shown to predict glaucomatous progression better than cells have wound up labeled as RGCs.99 functional and structural evaluations independently.82,85,86 Genetically encoded calcium indicators like GCaMPs are being explored as possible functional imaging tools to Artificial Intelligence and Telemedicine measure RGC damage.99 Fluorescently labeled GCaMP3 retinal cells can be detected in vivo with confocal Artificial Intelligence scanning laser ophthalmoscopy when directly exposed to ultraviolet light. Although originally explored in Artificial intelligence is a computerized method that can be transgenic mice, this labeling has now been performed trained to detect relationships between information from using viral vectors, paving the way for its development multiple inputs and an output of interest. This method has into a tool for imaging human eyes in vivo.100 been applied to visual function testing and optical imaging labeling with retrograde tracer mol- and has indicated an ability to diagnose and detect disease ecules, such as Fluorogold (Fluorochrome, LLC, Denver, change accurately over time.87 One approach of artificial CO), is highly effective in mouse models.99 Transgenic intelligence, namely deep learning, has gained popularity mouse models also have been used with a number of recently as a promising tool for sensitive detection of RGC-specific markers, such as THY monocyte differentia- disease progression. This method was developed to imitate tion antigen 1, ckit, and Brn3b.95 Transgenic THY monocyte multilevel processing performed by the human brain using differentiation antigen 1 mice, that have been modified to linear and nonlinear models. Deep learning has been express yellow fluorescent protein, permit detailed shown to be valuable in improving image quality,88,89 visualization of neurons within the RGC. After labeling, enhancing reliability of automated segmentation,90 and these can be visualized in vivo with confocal diagnosing glaucoma.91,92 scanning laser ophthalmoscopy. Unfortunately, however, specificity can be lost as labels are phagocytosed by Telemedicine microglia and macrophages, and in many cases, the tracer’s use is limited by its transient presence.99 Electroporation, The application of telemedicine has expanded throughout an in vivo application of voltage to a membrane for medicine; accordingly, in glaucoma care, most uses are in transfection of genes or contrast agents into cells, is another disease screening with optic nerve photographs transmitted to 93 labeling technique used in animal models that, although glaucoma specialists. The ease of acquiring multiple images very sensitive, is limited by its reduced specificity.101 Thus opens new opportunities for detection of disease progression. far, none of these methods can be applied to humans.99 In conclusion, ophthalmic imaging innovations have Novel Biomarkers for Glaucoma revolutionized glaucoma diagnosis and management, and Progression the tools, algorithms, and biomarkers that are relevant to clinical practice are evolving constantly. As these parame- ters undergo constant research and development, they may Direct RGC imaging offers exciting prospects for diagnostic contribute to further improvements in glaucoma progression imaging in humans. Detection of apoptosing retinal cells is detection. These novel biomarkers and approaches may aid one such method that has offered encouraging results thus in the understanding of specific causes and pathologic far. Annexin-5 is an endogenous extracellular calcium- mechanisms of glaucoma, such as those involving the dependent membrane-binding protein ubiquitously biomechanics of the LC, thus contributing to the develop- expressed in human cells. In an early step in apoptosis, ment of future new therapies. phosphatidylserine is translocated to the outer plasma membrane, where it is vulnerable to high-affinity binding with a fluorescently labeled annexin-5 molecule, which References subsequently can be visualized and counted using confocal scanning laser ophthalmoscopy.94,95 In a proof-of-concept 1. Quigley HA, Broman AT. The number of people with study, it was demonstrated that this method could be used glaucoma worldwide in 2010 and 2020. Br J Ophthalmol. effectively in human eyes in vivo. Interestingly, in their post 2006;90(3):262-267. hoc analysis, the authors found high detection of apoptosing 2. Bussel II, Wolstein G, Schuman JS. OCT for glaucoma retinal cell counts could predict increased glaucoma pro- diagnosis, screening and detection of glaucoma progression. gression rates.96 Phase 2 studies are being undertaken to Br J Ophthalmol. 2014;98(Suppl 2):ii15-ii19.

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Footnotes and Financial Disclosures

Originally received: July 4, 2017. Analysis and interpretation: Lavinsky, Wollstein, Tauber, Schuman Final revision: September 11, 2017. Data collection: Lavinsky, Wollstein, Tauber, Schuman Accepted: October 5, 2017. Manuscript no. 2017-391. Obtained funding: none NYU Langone Eye Center, New York University School of Medicine, New Overall responsibility: Lavinsky, Wollstein, Schuman York, New York. Financial Disclosure(s): Abbreviations and Acronyms: AO ¼ LC ¼ MRI ¼ The author(s) have made the following disclosure(s): J.S.S.: Royalties e adaptive optics; lamina cribrosa; magnetic resonance ONH ¼ POAG ¼ Zeiss imaging; optic nerve head; primary open-angle glau- coma; RGC ¼ retinal ganglion cell; RNFL ¼ retinal nerve fiber layer; Supported in part by the National Institutes of Health, Bethesda, Maryland SAP ¼ standard automated perimetry. (grant no.: R01-EY013178). HUMAN SUBJECTS: No human subjects were included in this study. Correspondence: Joel S. Schuman, MD, NYU Langone Eye Center, 462 First Avenue, NBV Author Contributions: 5N1, New York, NY 10016. E-mail: [email protected]. Conception and design: Lavinsky, Wollstein, Tauber, Schuman

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