Noninvasive, In Vivo Assessment of Mouse Retinal Structure Using Optical Coherence Tomography

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Abstract

Background

Optical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration.

Methodology/Principal Findings

We achieved to adapt a commercial 3 ^rd generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber's congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified.

Conclusions/Significance

We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies.

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Most cited references 16

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Optical coherence tomography for ultrahigh resolution in vivo imaging.

James G Fujimoto (2003)

Optical coherence tomography (OCT) is an emerging biomedical optical imaging technique that performs high-resolution, cross-sectional tomographic imaging of microstructure in biological systems. OCT can achieve image resolutions of 1-15 microm, one to two orders of magnitude finer than standard ultrasound. The image penetration depth of OCT is determined by the optical scattering and is up to 2-3 mm in tissue. OCT functions as a type of 'optical biopsy' to provide cross-sectional images of tissue structure on the micron scale. It is a promising imaging technology because it can provide images of tissue in situ and in real time, without the need for excision and processing of specimens.

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Molecular mechanisms of light-induced photoreceptor apoptosis and neuroprotection for retinal degeneration.

Andreas Wenzel, Christian Grimm, Marijana Samardzija … (2005)

Human retinal dystrophies and degenerations and light-induced retinal degenerations in animal models are sharing an important feature: visual cell death by apoptosis. Studying apoptosis may thus provide an important handle to understand mechanisms of cell death and to develop potential rescue strategies for blinding retinal diseases. Apoptosis is the regulated elimination of individual cells and constitutes an almost universal principle in developmental histogenesis and organogenesis and in the maintenance of tissue homeostasis in mature organs. Here we present an overview on molecular and cellular mechanisms of apoptosis and summarize recent developments. The classical concept of apoptosis being initiated and executed by endopeptidases that cleave proteins at aspartate residues (Caspases) can no longer be held in its strict sense. There is an increasing number of caspase-independent pathways, involving apoptosis inducing factor, endonuclease G, poly-(ADP-ribose) polymerase-1, proteasomes, lysosomes and others. Similarly, a considerable number and diversity of pro-apoptotic stimuli is being explored. We focus on apoptosis pathways in our model: light-damage induced by short exposures to bright white light and highlight those essential conditions known so far in the apoptotic death cascade. In our model, the visual pigment rhodopsin is the essential mediator of the initial death signal. The rate of rhodopsin regeneration defines damage threshold in different strains of mice. This rate depends on the level of the pigment epithelial protein RPE65, which in turn depends on the amino acid (leucine or methionine) encoded at position 450. Activation of the pro-apoptotic transcription factor AP-1 constitutes an essential death signal. Inhibition of rhodopsin regeneration as well as suppression of AP-1 confers complete protection in our system. Furthermore, we describe observations in other light-damage systems as well as characteristics of animal models for RP with particular emphasis on rescue strategies. There is a vast array of different neuroprotective cytokines that are applied in light-damage and RP animal models and show diverging efficacy. Some cytokines protect against light damage as well as against RP in animal models. At present, the mechanisms of neuroprotective/anti-apoptotic action represent a "black box" which needs to be explored. Even though acute light damage and RP animal models show different characteristics in many respects, we hope to gain insights into apoptotic mechanisms for both conditions by studying light damage and comparing results with those obtained in animal models. In our view, future directions may include the investigation of different apoptotic pathways in light damage (and inherited animal models). Emphasis should also be placed on mechanisms of removal of dead cells in apoptosis, which appears to be more important than initially recognized. In this context, a stimulating concept concerns age-related macular degeneration, where an insufficiency of macrophages removing debris that results from cell death and photoreceptor turnover might be an important pathogenetic event. In acute light damage, the appearance of macrophages as well as phagocytosis by the retinal pigment epithelium are a consistent and conspicuous feature, which lends itself to the study of removal of cellular debris in apoptosis. We are aware of the many excellent reviews and the earlier work paving the way to our current knowledge and understanding of retinal degeneration, photoreceptor apoptosis and neuroprotection. However, we limited this review mainly to work published in the last 7-8 years and we apologize to all the researchers which have contributed to the field but are not cited here.

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Crumbs homologue 1 is required for maintenance of photoreceptor cell polarization and adhesion during light exposure.

Mathias Seeliger, Albena Kantardzhieva, Inge Versteeg … (2004)

Loss of Crumbs homologue 1 (CRB1) function causes either the eye disease Leber congenital amaurosis or progressive retinitis pigmentosa, depending on the amount of residual CRB1 activity and the genetic background. Crb1 localizes specifically to the sub-apical region adjacent to the adherens junction complex at the outer limiting membrane in the retina. We show that it is associated here with multiple PDZ protein 1 (Mupp1), protein associated with Lin-7 (Pals1 or Mpp5) and Mpp4. We have produced Crb1(-/-) mice completely lacking any functional Crb1. Although the retinas are initially normal, by 3-9 months the Crb1(-/-) retinas develop localized lesions where the integrity of the outer limiting membrane is lost and giant half rosettes are formed. After delamination of the photoreceptor layer, neuronal cell death occurs in the inner and outer nuclear layers of the retina. On moderate exposure to light for 3 days at 3 months of age, the number of severe focal retinal lesions significantly increases in the Crb1(-/-) retina. Crb2, Crb3 and Crb1 interacting proteins remain localized to the sub-apical region and therefore are not sufficient to maintain cell adhesion during light exposure in Crb1(-/-) retinas. Thus we propose that during light exposure Crb1 is essential to maintain, but not assemble, adherens junctions between photoreceptors and Müller glia cells and prevents retinal disorganization and dystrophy. Hence, light may be an influential factor in the development of the corresponding human diseases.

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Author and article information

Contributors

: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2009

Publication date (Electronic): 19 October 2009

Volume: 4

Issue: 10

Electronic Location Identifier: e7507

Affiliations

[1 ]Division of Ocular Neurodegeneration, Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tuebingen, Tuebingen, Germany

[2 ]Institute of Animal Welfare, Ethology and Animal Hygiene, Faculty of Veterinary Medicine, Ludwig-Maximilians-University, Munich, Germany

[3 ]Laboratory of Retinal Cell Biology, University of Zurich, Zurich, Switzerland

[4 ]Neuromedical Genetics, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands

[5 ]Toronto Western Research Institute, University Health Network, Departments of Ophthalmology and Visual Science, and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

Vrije Universiteit Amsterdam, Netherlands

Author notes

* E-mail: see@ 123456uni-tuebingen.de

Conceived and designed the experiments: MDF MWS. Performed the experiments: MDF GH SB NT RM EF CG AW CER SAvdP JW MWS. Analyzed the data: MDF GH SB NT RM EF CG AW CER SAvdP JW MP RB MWS. Contributed reagents/materials/analysis tools: MDF GH SB NT RM EF CG AW CER SAvdP JW MP RB MWS. Wrote the paper: MWS. Edited the manuscript: CG AW CER SAvdP JW MP RB.

[¤a]

Current address: Novartis Pharma Schweiz AG, Berne, Switzerland

[¤b]

Current address: VU University Medical Center, Department of Molecular Cell Biology and Immunology, Amsterdam, The Netherlands

Article

Publisher ID: 09-PONE-RA-10706R1

DOI: 10.1371/journal.pone.0007507

PMC ID: 2759518

PubMed ID: 19838301

SO-VID: 486d9ae1-2be8-4717-86dc-d46cc12ef342

Copyright © Fischer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 31 May 2009

Date accepted : 21 September 2009

Page count

Pages: 7

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