Aaron Nagiel

Specificity of afferent synapses onto plane-polarized hair cells in the posterior lateral line of the zebrafish

The proper wiring of the vertebrate brain represents an extraordinary developmental challenge, requiring billions of neurons to select their appropriate synaptic targets. In view of this complexity, simple vertebrate systems provide necessary models for understanding how synaptic specificity arises. The posterior lateral-line organ of larval zebrafish consists of polarized hair cells organized in discrete clusters known as neuromasts. Here we show that each afferent neuron of the posterior lateral line establishes specific contacts with hair cells of the same hair-bundle polarity. We quantify this specificity by modeling the neuron as a biased selector of hair-cell polarity and find evidence for bias from as early as 2.5 d after fertilization. More than half of the neurons form contacts on multiple neuromasts, but the innervated organs are spatially consecutive and the polarity preference is consistent. Using a novel reagent for correlative electron microscopy, HRP-mCherry, we show that these contacts are indeed afferent synapses bearing vesicle-loaded synaptic ribbons. Moreover, afferent neurons reassume their biased innervation pattern after hair-cell ablation and regeneration. By documenting specificity in the pattern of neuronal connectivity during development and in the context of organ regeneration, these results establish the posterior lateral-line organ as a vertebrate system for the in vivo study of synaptic target selection.

A Promising Future for Optical Coherence Tomography Angiography.

Fluorescein angiography has been the gold standard imaging modality for the retinal vasculature since its groundbreaking introduction in 1961 by Alvis and Novotny and has revolutionized our ability to diagnose diseases of the retinal vasculature and to identify retinal and choroidal neovascularization.1 Its greatest advantage may lie in its ability to detect dynamic patterns of dye transit and leakage. Various innovations have broadened the use of angiography, including the application of indocyanine green, wide-field image acquisition, confocal scanning laser ophthalmoscopy, and adaptive optics. However, a major limitation of traditional angiography resides in its inability to image the entire retinal capillary system or to directly visualize nascent vessels, leaving the practitioner to deduce the presence of neovascularization on the basis of other indicators such as fluid, leakage, or edema. Optical coherence tomography (OCT) angiography applies high-speed OCT scanning to detect blood flow by analyzing signal decorrelation between scans. Compared with stationary areas of the retina, the movement of erythrocytes within a vessel generates a decorrelated signal. The split-spectrum amplitude decorrelation angiography (SSADA) algorithm improves the signal to noise ratio by splitting the source spectrum into 4 parts and averaging the resultant 4 signals. High-density raster scanning of a 2-dimensional area of the retina generates a volumetric rendering of blood flow from the internal limiting membrane to the choroid and allows for direct visualization of normal and abnormal blood vessels. The currently available Optovue AngioVue System uses spectral-domain technology, an 840-nm laser, and the SSADA algorithm (Figure). The 70-kHz A-scan rate on this device allows a 3 × 3-mm OCT angiography volume to be acquired in 3 seconds. The lateral and axial resolutions are both 15 μm; the axial resolution is significantly less than that for structural OCT (5 μm) owing to signal averaging. Another device under development by Zeiss will feature a swept-source laser centered at 1040 nm with the potential for augmented signal penetration depth. Future devices may use other approaches for OCT angiography, such as phase contrast or intensity variance. Unlike traditional angiography, which uses a fluorescent dye and provides limited 3-dimensional information, OCT angiography requires no exogenous contrast and uses dense volumetric scanning to provide depth-resolved visualization of the retinal and choroidal vasculature. The retinal vasculature of the central macula is predominantly a 3-layered capillary bed. Even though one can identify the superficial retinal capillary plexus using fluorescein angiography, this angiographic technique poorly visualizes the intermediate and deep plexuses that are a critical focus of retinal vascular disease.1,2 Using the SSADA technique, Spaide et al1 were the first to demonstrate distinct superficial and deep capillary networks, the latter of which includes both the deep and intermediate plexuses. The use of OCT angiography could greatly enrich our understanding of the ischemic processes affecting different layers of the retinal vasculature, such as cotton-wool spots (superficial plexus ischemia), paracentral acute middle maculopathy (deep plexus ischemia),2 and macular telangiectasia type 2.3 Optical coherence tomography angiography may also have the potential to dissect the long-debated origin and microvascular anatomy of neovascularization in age-related macular degeneration, including type 1 (sub– retinal pigment epithelium), type 2 (subretinal), and type 3 (intraretinal; retinal angiomatous proliferation) neovascularization. In a seminal study, Jia et al4 generated 3-dimensional reconstructions of choroidal neovascularization and en face OCT renderings to highlight the precise vascular anatomy of choroidal neovascularization. It may be possible to identify distinct morphologies of choroidal neovascularization and then correlate these subtypes with disease course, prognosis, and response to treatment. In addition to providing enhanced anatomic detail, OCT angiography intrinsically generates data on vascular flow. This powerful feature has enormous implications for understanding tissue perfusion in the absence of obvious morphological changes. A flow index of the optic nerve head can be used to ascertain disc perfusion. For example, glaucomatous optic discs and discs damaged by optic neuritis have significantly diminished flow indices compared with normal discs.5,6 Remarkably, OCT angiographic measurements are sensitive

Mechanism of Retinal Pigment Epithelium Tear Formation Following Intravitreal Anti–Vascular Endothelial Growth Factor Therapy Revealed by Spectral-Domain Optical Coherence Tomography

PURPOSE To demonstrate the mechanism by which retinal pigment epithelium (RPE) tears occur in eyes with neovascular age-related macular degeneration (AMD) treated with intravitreal anti-vascular endothelial growth factor (VEGF) agents using spectral-domain optical coherence tomography (OCT). DESIGN Retrospective observational case series. METHODS OCT images of 8 eyes that developed RPE tears following the administration of intravitreal anti-VEGF agents for neovascular AMD were evaluated. Pretear and posttear images were compared in order to elucidate the mechanism by which RPE tears occur in this setting. RESULTS In all eyes, pretear images revealed a vascularized pigment epithelial detachment (PED) containing hyperreflective material consistent with choroidal neovascularization (CNV). This CNV was adherent to the undersurface of the RPE and created contractile folds in the RPE contour. In 6 eyes, contractile neovascular tissue spanned the PED, causing outward bowing of the Bruch membrane and a peaked appearance to the overlying RPE monolayer. RPE tears occurred after the first anti-VEGF injection in 6 of 8 eyes. The posttear OCT images showed a discontinuity in the RPE with the CNV adherent to the retracted RPE. In all eyes, the RPE ruptured along a segment of bare RPE not in contact with the CNV or Bruch membrane. CONCLUSIONS Eyes with vascularized PEDs secondary to AMD may show specific OCT findings that increase the risk for RPE tear following intravitreal anti-VEGF injection. Rapid involution and contraction of neovascular tissue adherent to the undersurface of the RPE may impart a substantial contractile force that tears this already-strained tissue layer.

Type 3 neovascularization: evolution, association with pigment epithelial detachment, and treatment response as revealed by spectral domain optical coherence tomography.

Purpose: To demonstrate the evolution and treatment response of Type 3 neovascularization using spectral domain optical coherence tomography. Methods: We retrospectively analyzed 40 eyes treated with intravitreal anti-vascular endothelial growth factor therapy for Type 3 neovascularization over a variable follow-up period. Results: In 17 eyes, spectral domain optical coherence tomography captured the development of Type 3 neovascularization from punctate hyperreflective foci that preceded any outer retinal defect. The more mature Type 3 lesions were associated with outer retinal disruption and adjacent cystoid macular edema. In addition, 37 of 40 Type 3 lesions (93%) were associated with an underlying pigment epithelial detachment, of which 26 (70%) were drusenoid, 6 (16%) serous, and 5 (14%) mixed. Type 3 vessels appeared to leak fluid into the pigment epithelial detachment cavity, creating serous pigment epithelial detachments as large as 925 &mgr;m in maximal height. Treatment with anti-vascular endothelial growth factor agents led to prompt involution of the lesion and resorption of the intraretinal and subretinal pigment epithelium fluid after one or two injections (median = 1). Conclusion: In some eyes with age-related macular degeneration, the earliest sign of Type 3 neovascularization is punctate hyperreflective foci above the external limiting membrane. The mature Type 3 lesions and associated serous pigment epithelial detachments are highly responsive to anti-vascular endothelial growth factor therapy.

The transmembrane inner ear (Tmie) protein is essential for normal hearing and balance in the zebrafish

Little is known about the proteins that mediate mechanoelectrical transduction, the process by which acoustic and accelerational stimuli are transformed by hair cells of the inner ear into electrical signals. In our search for molecules involved in mechanotransduction, we discovered a line of deaf and uncoordinated zebrafish with defective hair-cell function. The hair cells of mutant larvae fail to incorporate fluorophores that normally traverse the transduction channels and their ears lack microphonic potentials in response to vibratory stimuli. Hair cells in the posterior lateral lines of mutants contain numerous lysosomes and have short, disordered hair bundles. Their stereocilia lack two components of the transduction apparatus, tip links and insertional plaques. Positional cloning revealed an early frameshift mutation in tmie, the zebrafish ortholog of the mammalian gene transmembrane inner ear. The mutant line therefore affords us an opportunity to investigate the role of the corresponding protein in mechanoelectrical transduction.

Subretinal Transplantation of Embryonic Stem Cell-Derived Retinal Pigment Epithelium for the Treatment of Macular Degeneration: An Assessment at 4 Years.

Advanced macular degeneration is an important cause of vision loss in the United States with over 2 million people affected by the disease. Despite substantial progress in the development of new therapies for wet AMD, the severe visual impairment associated with geographic atrophy in dry AMD or Stargardt disease remains untreatable. Recently, two phase I/II studies involving 18 patients with these diseases have demonstrated that it is possible to safely implant human embryonic stem cell-derived RPE (hESC-RPE) in an attempt to rescue photoreceptors and visual function. The anatomical and functional results are encouraging, with more than half of treated patients experiencing sustained improvements in visual acuity and demonstrating evidence of possible cellular engraftment. However, any conclusions remain tempered by the relatively short follow-up time, lack of a formal control group, poor initial visual acuity, and small number of patients. Aside from an instance of postoperative infectious endophthalmitis, no adverse events related to the cell therapy, such as hyperproliferation, tumorigenicity, or rejection-related inflammation were noted in this initial cohort of 18 patients. These first-in-human safety studies have opened the door to future studies enrolling patients with less advanced disease, treating other diseases that result in RPE loss, employing shorter immunosuppressive regimens, and using alternative strategies for RPE transplantation such as sheets of cells with or without scaffolding to mimic Bruchs membrane. The ultimate goal of these initial safety studies is to promote continued translation of complex biological therapies into meaningful treatment strategies that may address unmet medical needs.

ULTRA-WIDEFIELD FUNDUS IMAGING: A Review of Clinical Applications and Future Trends.

Purpose: To review the basic principles of ultra-widefield fundus imaging and discuss its clinical utility for a variety of retinal and choroidal disorders. Methods: A systematic review of the PubMed database was performed using the search terms Optos, optomap, panoramic, ultra-widefield, wide-angle, and ellipsoid mirror. This yielded 158 publications of which 128 were selected based on content and relevance. Results: A total of 128 articles pertaining to ultra-widefield imaging were cited in this review. Conclusion: Optos ultra-widefield imaging has become an essential tool for the identification of peripheral retinal and vascular pathology. The high resolution and multimodal capabilities of this device are also providing new insights into a variety of disorders, even those that primarily involve the posterior pole. Although the presence of artifact and the need for clinical validation are significant hurdles to more widespread use, ultra-widefield is evolving to become the standard-of-care imaging modality for many diseases and is finding new clinical and research applications such as for screening and telemedicine.

Activity-independent specification of synaptic targets in the posterior lateral line of the larval zebrafish

The development of functional neural circuits requires that connections between neurons be established in a precise manner. The mechanisms by which complex nervous systems perform this daunting task remain largely unknown. In the posterior lateral line of larval zebrafish, each afferent neuron forms synaptic contacts with hair cells of a common hair-bundle polarity. We investigated whether afferent neurons distinguish hair-cell polarities by analyzing differences in the synaptic signaling between oppositely polarized hair cells. By examining two mutant zebrafish lines with defects in mechanoelectrical transduction, and by blocking transduction during the development of wild-type fish, we found that afferent neurons could form specific synapses in the absence of stimulus-evoked patterns of synaptic release. Asking next whether this specificity arises through intrinsically generated patterns of synaptic release, we found that the polarity preference persisted in two mutant lines lacking essential synaptic proteins. These results indicate that lateral-line afferent neurons do not require synaptic activity to distinguish hair-cell polarities and suggest that molecular labels of hair-cell polarity guide prepatterned afferents to form the appropriate synapses.

RESOLUTION OF A GIANT PIGMENT EPITHELIAL DETACHMENT WITH HALF-DOSE AFLIBERCEPT.

PURPOSE To describe the use of half-dose anti-vascular endothelial growth factor therapy in a patient with giant pigment epithelial detachments. METHODS Observational case report. A 76-year-old woman with neovascular age-related macular degeneration presented with massive bilateral pigment epithelial detachments measuring over 1000 μm in height. Her right eye was treated with standard-dose aflibercept, which led to two large retinal pigment epithelium tears. Treatment of the left eye with half-dose aflibercept led to complete resolution of the detachment without tear formation. RESULTS Half-dose anti-vascular endothelial growth factor therapy resulted in resolution of a giant pigment epithelial detachment ∼1500 μm in maximal height and 10 mm in diameter. CONCLUSION Reduced-dose anti-vascular endothelial growth factor therapy may be considered as a treatment option for very large pigment epithelial detachments at high risk for retinal pigment epithelium tear formation.

Isolated Zygomycetes Endophthalmitis: A Case Report.

Mucormycosis is a rare fungal infection typically limited to immunocompromised patients or patients with diabetes., Clinical manifestation varies, with rhino-orbital-cerebral mucormycosis as the most common presentation. The authors present here a 56-year-old man who complained of isolated OD pain with no evidence of cerebral or sinus inflammation on imaging. Enucleation was eventually performed, and histopathology of the globe demonstrated characteristics of zygomycetes infection. Intraocular mucormycosis is rare and when reported is related to contiguous spread of disease or surgical inoculation. After thorough literature review, the authors believe this to be the first reported case of isolated intraocular mucormycosis in a patient with no prior history of ocular surgery.