Glenn Yiu

Glial inhibition of CNS axon regeneration

Damage to the adult CNS often leads to persistent deficits due to the inability of mature axons to regenerate after injury. Mounting evidence suggests that the glial environment of the adult CNS, which includes inhibitory molecules in CNS myelin as well as proteoglycans associated with astroglial scarring, might present a major hurdle for successful axon regeneration. Here, we evaluate the molecular basis of these inhibitory influences and their contributions to the limitation of long-distance axon repair and other types of structural plasticity. Greater insight into glial inhibition is crucial for developing therapies to promote functional recovery after neural injury.

Protecting Axonal Degeneration by Increasing Nicotinamide Adenine Dinucleotide Levels in Experimental Autoimmune Encephalomyelitis Models

Axonal damage is a major morphological alteration in the CNS of patients with multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the underlying mechanism for the axonal damage associated with MS/EAE and its contribution to the clinical symptoms remain unclear. The expression of a fusion protein, named “Wallerian degeneration slow” (Wlds), can protect axons from degeneration, likely through a β-nicotinamide adenine dinucleotide (NAD)-dependent mechanism. In this study, we find that, when induced with EAE, Wlds mice showed a modest attenuation of behavioral deficits and axon loss, suggesting that EAE-associated axon damage may occur by a mechanism similar to Wallerian degeneration. Furthermore, nicotinamide (NAm), an NAD biosynthesis precursor, profoundly prevents the degeneration of demyelinated axons and improves the behavioral deficits in EAE models. Finally, we demonstrate that delayed NAm treatment is also beneficial to EAE models, pointing to the therapeutic potential of NAm as a protective agent for EAE and perhaps MS patients.

Signaling mechanisms of the myelin inhibitors of axon regeneration.

One of the major obstacles to successful axon regeneration in the adult CNS is the presence of inhibitory molecules that are associated with myelin. Recent studies have identified several major myelin-associated inhibitors along with the relevant signaling molecules. Such advances have not only enhanced our understanding of the signaling mechanisms that are involved in the inhibition of axon regeneration in the adult CNS but also allowed us to assess the therapeutic potential of blocking these inhibitory influences to promote axon regeneration.

Retrograde BMP Signaling Regulates Trigeminal Sensory Neuron Identities and the Formation of Precise Face Maps

Somatosensory information from the face is transmitted to the brain by trigeminal sensory neurons. It was previously unknown whether neurons innervating distinct areas of the face possess molecular differences. We have identified a set of genes differentially expressed along the dorsoventral axis of the embryonic mouse trigeminal ganglion and thus can be considered trigeminal positional identity markers. Interestingly, establishing some of the spatial patterns requires signals from the developing face. We identified bone morphogenetic protein 4 (BMP4) as one of these target-derived factors and showed that spatially defined retrograde BMP signaling controls the differential gene expressions in trigeminal neurons through both Smad4-independent and Smad4-dependent pathways. Mice lacking one of the BMP4-regulated transcription factors, Onecut2 (OC2), have defects in the trigeminal central projections representing the whiskers. Our results provide molecular evidence for both spatial patterning and retrograde regulation of gene expression in sensory neurons during the development of the somatosensory map.

Characterization of the Choroid-Scleral Junction and Suprachoroidal Layer in Healthy Individuals on Enhanced-Depth Imaging Optical Coherence Tomography

IMPORTANCE Accurate measurements of choroidal thickness (CT) using enhanced-depth imaging optical coherence tomography (EDI-OCT) require a well-defined choroid-scleral junction (CSJ), which may appear in some eyes as a hyporeflective band corresponding to the suprachoroidal layer (SCL). OBJECTIVE To identify factors associated with the presence and thickness of the SCL in healthy participants and determine how different CSJ boundary definitions impact CT measurements. DESIGN, SETTING, AND PARTICIPANTS Secondary analysis of EDI-OCT images obtained prospectively from 74 eyes of 74 controls (mean age, 68.6 years) from the Age-Related Eye Disease Study 2 Ancillary SDOCT Study. MAIN OUTCOMES AND MEASURES The CSJ appearances were categorized as either having no visible SCL or a hyporeflective band corresponding to the SCL. Ocular parameters associated with the presence and thickness of the SCL were identified. Subfoveal CT was measured using 3 different posterior boundaries: (1) the posterior vessel border (vascular CT [VCT]), (2) inner border of the SCL (stromal CT [StCT]), and (3) inner border of the sclera (total CT [TCT]). Manual segmentation using custom software was used to compare VCT, StCT, and TCT across the macula. RESULTS The SCL was visible in 33 eyes (44.6%). Factors associated with SCL presence and thickness included hyperopic refractive error (R2 = 0.123; P = .045) and increased TCT (R2 = 0.215; P = .004), but not age, visual acuity, intraocular pressure, retinal foveal thickness, VCT, or StCT. In eyes where the SCL was not visible, mean [SD] subfoveal VCT was 222.3 [101.5] μm and StCT and TCT were 240.0 [99.0] μm, with a difference of 17.7 [16.0] μm (P < .001). In eyes where the SCL was visible, mean [SD] subfoveal VCT, StCT, and TCT were 221.9 [83.1] μm, 257.7 [97.3] μm, and 294.1 [104.8] μm, respectively, with the greatest difference of 72.2 [30.4] μm between VCT and TCT (P < .001). All 3 CT measurements were significantly different along all points up to 3.0 mm nasal and temporal to the fovea. CONCLUSIONS AND RELEVANCE A hyporeflective SCL is visible at the CSJ on EDI-OCT in nearly half of healthy individuals, and its presence correlates with hyperopia. Different posterior boundary definitions may result in significant differences in CT measurements and should be explicitly identified in future choroidal studies and segmentation algorithms.

Effect of Anti–Vascular Endothelial Growth Factor Therapy on Choroidal Thickness in Diabetic Macular Edema

PURPOSE To determine the effect of anti-vascular endothelial growth factor (VEGF) therapy on choroidal thickness in eyes with diabetic macular edema (DME). DESIGN A retrospective, cohort analysis of 59 eyes from 59 patients with DME without prior anti-VEGF therapy. METHODS Choroidal thickness was measured using semiautomated segmentation of enhanced depth imaging optical coherence tomography images at 0.5-mm intervals from 2.5 mm nasal to 2.5 mm temporal to the fovea. Changes in choroidal thickness with and without anti-VEGF treatment over 6 months were compared. Best-corrected visual acuity and central foveal thickness were analyzed to evaluate the association of choroidal thickness with functional and anatomic outcomes. RESULTS Of the 59 eyes with DME, 26 eyes were observed without treatment, whereas 33 underwent intravitreal anti-VEGF therapy (mean number of injections, 2.73) over 6 months. In untreated eyes, there was no significant change in best-corrected visual acuity (P = .098), central foveal thickness (P = .472), or choroidal thickness at all measurements along the macula (P = .057 at the fovea). In eyes treated with anti-VEGF injections, choroidal thickness decreased significantly at the fovea (246.6 to 224.8 μm; P < .001) and at 0.5 mm nasal (240.9 to 221.9 μm; P = .002) and 0.5 mm temporal (249.3 to 224.8 μm; P = .011) to the fovea. The decrease in subfoveal choroidal thickness after anti-VEGF treatment was not associated with the cumulative number of anti-VEGF injections (R(2) = 0.031; P = .327) or to changes in best-corrected visual acuity (R(2) = 0.017; P = .470) or central foveal thickness (R(2) = 0.040; P = .263). CONCLUSIONS Central choroidal thickness decreases after anti-VEGF therapy for DME after 6 months, but may not be associated with functional or anatomic outcomes in eyes with DME.

Relationship of Central Choroidal Thickness With Age-Related Macular Degeneration Status

PURPOSE To compare choroidal thickness in patients with intermediate or advanced age-related macular degeneration (AMD) and control subjects using enhanced-depth imaging optical coherence tomography (EDI-OCT). DESIGN Retrospective cross-sectional study of 325 eyes from 164 subjects who underwent EDI-OCT for the Age-Related Eye Disease Study (AREDS) 2 Ancillary Spectral Domain OCT study. METHODS Choroidal thickness was measured by semi-automated segmentation of EDI-OCT images from 1.5 mm nasal to 1.5 mm temporal to the fovea. Multivariate linear regression was used to evaluate the association of subfoveal choroidal thickness or average choroidal thickness across the central 3-mm segment with systemic and ocular variables. Choroidal thickness measurements were compared between eyes with no AMD (n = 154) (ie, controls), intermediate AMD (n = 109), and advanced AMD (n = 62). RESULTS Both subfoveal and average choroidal thicknesses were associated with age (P < .001) and refractive error (P < .001), but not other variables tested. Mean average choroidal thickness was significantly reduced in advanced AMD as compared with control eyes (P = .008), with no significant difference between advanced and intermediate AMD eyes (P = .152) or between intermediate AMD and control eyes (P = .098). Choroidal thinning was also noted from 1.5 mm nasal to 1.5 mm temporal to the fovea when comparing advanced AMD with control eyes (P < .05 at all 0.5 mm interval locations). After adjustment for age and refractive error, however, there was no significant difference in subfoveal (P = .675) or average choroidal thickness (P = .746) across all 3 groups. CONCLUSIONS When adjusted for age and refractive error, central choroidal thickness may not be significantly influenced by AMD status based on AREDS categorization.

Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.

Purpose To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. Methods CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcus pyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay. Results Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected. Conclusions The CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.

Ocular Safety of Recreational Lasers

High-powered recreational lasers with the potential to cause severe ocular injuries are becoming increasingly available to the general public. Recently, a 9-yearold boy presented to our clinic with bilateral vision loss after playing with an adult who directed a handheld laser into both of his eyes. Known as the Spyder III Pro Arctic, the device was a class 4, high-powered 1250 mW laser that is manufactured from the 445 nm blue diode of a dismantled home theater projector and that is commercially available for online purchase from overseas. On initial examination, the patient’s vision was 20/126 in the right eye and 20/100 in the left. Dilated funduscopic examination revealed preretinal hemorrhages in the macula of both eyes (Figure, A). Cross-sectional images obtained by spectral-domain optical coherence tomography demonstrated both hemorrhages to be subhyaloidor subinternal-limiting membrane in location (Figure, B). Fortunately, the patient’s vision gradually returned to 20/20 in the left eye after a week and to 20/25 in the right eye after 2 months, with corresponding improvement in the preretinal hemorrhages in both eyes (Figure, C). Fundus autofluorescence images revealed no evidence of damage to the retinal pigment epithelium or Bruch’s membrane (Figure, D). Ever since the first laser was developed in the 1960s, laser products have permeated society with a wide variety of applications in military, industrial, laboratory, medical, and recreational settings. However, the expanding use of lasers in everyday life also increases the risk of injuries associated with laser exposure. Because light radiation in the visible and near-infrared portion of the electromagnetic spectrum is focused by the refractive structures of the human eye onto the retina, the irradiance onto the retinal surface is amplified by 5 to 6 orders of magnitude, making the retina the ocular tissue most vulnerable to laser injuries. Natural protective responses such as the blink reflex, pupillary constriction, and aversive head-turn response typically minimize sustained ocular exposure but do not prevent accidental laser eye injuries from occurring. Most reported cases of laser injuries occur in occupational environments, primarily with pulsed lasers in industrial or laboratory settings.1 The severity depends on the characteristics of the laser (wavelength, energy, continuous vs pulsed emission) and conditions of exposure (duration, distance, angle of incidence). For pulsed neodymium-doped yttrium aluminum garnet lasers, which account for many reported laser injuries, threshold studies in monkeys measured the median effective dose, the energy level required to produce a lesion in 50% of cases, to be 1.7 to 2.3 mJ for subretinal and vitreous hemorrhages caused by damage to choroidal vessels and to be less than 7 μJ for intraretinal hemorrhages arising from retinal vessel injury.2 These studies form the basis for laser exposure limits in humans, also known as the maximum permissible exposure limits, which are delineated in the American National Standards Institute’s laser safety standards.3 These parameters guide the classification of laser devices and determine how they must be labeled or what safety features are required. In contrast to lasers in industrial or laboratory settings, those available to consumers in the past were handheld red diode or helium-neon lasers with an output power of 1 to 5 mW. Retinal damage is unlikely to occur at such low output powers under most circumstances. Modern advancements in laser technology, however, have led to the commercial availability of powerful blue and green lasers. These shorter wavelength lasers, while helpful in such applications as increasing optical data storage in Blu-Ray technology, are much better absorbed by melanin in the retinal pigment epithelium and thus have a greater potential for ocular damage.4 Despite government restrictions, highpowered continuous-wave lasers, some in excess of 1250 mW, are becoming readily available to consumers via overseas vendors on the Internet. Furthermore, these high-powered handheld lasers may be difficult to distinguish from less harmful laser pointers. Teenagers in 2 recent case reports suffered bilateral retinal injuries while playing with “laser pointers,” one of which had a power of 150 mW.5,6 Unlike trained personnel in occupational settings, the public is generally uninformed about laser safety practices. In the case presented herein, the adult directed the laser at the child’s eyes in jest, unaware of the harmful consequences. Ocular injuries from consumer lasers are also underreported,7 further increasing the challenge to appropriately refine safety standards and regulations. Clinicians should be aware of the signs and symptoms of ocular laser injuries. Vision loss usually occurs immediately after laser exposure. It is often preceded by the perception of a bright flash and occasionally accompanied by an audible popping sound.8 Any pain is more likely the result of a self-inflicted corneal abrasion from eye rubbing than from actual retinal injury. Most laser injuries occur over short distances and lead to unilateral damage, although binocular injury can occur in outdoor settings due to beam divergence over long distances. Early evaluation by dilated funduscopic examination and ancillary imaging helps to determine whether visual complaints are consistent with any retinal abnormalities and provides documentation for medicolegal purposes. Ophthalmoscopy in the acute setting may show hemorrhages at various levels, depending on the location of the blood vessels damaged by the laser burn. In this child’s case, imaging studies suggest that the laser damage was limited to superficial retinal vessels with no involvement of the underlying retinal pigment VIEWPOINT

Current and investigational pharmacotherapeutic approaches for modulating retinal angiogenesis

Retinal vascular development is a carefully orchestrated developmental process during which retinal and choroidal vasculature form to provide a dual vascular supply to the neurosensory retina and retinal pigment epithelium. The most common causes of vision loss in children and adults involve at least in part perturbation of the normal vascular physiology or development. Vascular endothelial growth factor has emerged as a key molecular regulator of retinal vascular development as well as retinal and choroidal neovascularization, which underlie the pathophysiology of many retinal diseases. Over the past decade, the advent of injectable pharmacotherapeutic agents into the vitreous cavity of the eye has revolutionized our management of neovascular age-related macular degeneration and other retinal diseases and has, for the first time, offered an opportunity to improve vision rather than just slow the progression of disease processes. The transient duration of these agents, however, requires chronic treatment with repeated intraocular injections and significant treatment burden for patients and the healthcare system. Novel treatments modulating retinal angiogenesis offer the promise of improved efficacy, decreased treatment burden and improved cost–effectiveness.