Noninvasive, in vivo assessment of mouse retinal structure using optical coherence tomography

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Principle of Optical Coherence Tomography (OCT) and its application in rodents.

A) Schematic diagram of spectral-domain (SD) OCT. Green arrows indicate efferent, red arrows afferent light. B) Comparison of OCT to conventional ophthalmic imaging techniques. Top left: Conventional techniques either yield surface images of the retina (“fundus”), or in case of Scanning-Laser Ophthalmoscopy (SLO), confocal horizontal sections. Top right: Example of an SLO image of the central murine retina. Bottom left: In contrast, the OCT provides high-resolution vertical sections. Bottom right: Example of an OCT slice from the central murine retina in comparison to matching standard light microscopy. C) Representation of retinal layers in OCT and histology. See text for details. D) OCT recording setup for rodents. The schematic drawing of a mouse marks the recording position on the XYZ table. The eye is directly facing the OCT recording unit with a 78 dpt. lens attached.

Figure 2. OCT and histological morphometric data in rodents.

Relationship between OCT and histological morphometry in C57/BL6 mice broken down to retinal layers. A) Correlation of histological and OCT data. Pearson's correlation coefficient (R2) is based on the data of all quantified retinal layers. B) Comparison of retinal layer thickness between histology (dark) and OCT (light). There was no statistically significant difference between histological analysis and OCT-based quantification in any retinal layer using Student's t-test at a significance level of p<0.05. All data are reported as mean values±standard deviation (error bars).

Figure 3. OCT assessment of light-induced murine retinal degeneration.

A) OCT section across the central retina, containing adjacent damaged and non-damaged areas. B) Demarcation of the damaged area in vivo by SLO autofluorescence (AF) imaging based on fluorescent photoreceptor debris (marked by an asterisk). C) Detail of the transition zone between damaged and non-damaged retina in a). The asterisk marks the damaged area as in B). D), E) Comparison of the representation of light-induced retinal damage in histology and OCT. The arrowhead in the OCT image points towards a site of retinal edema.

Figure 4. Capability of the OCT to detect and capture the nature of lesions.

A)–F) Multiple retinal rosette formation in the neural retinal leucine zipper (Nrl) knockout mouse. A) SLO surface image (514 nm) in which the retinal rosettes (arrow) show as whitish dots. B) SLO autofluorescence image indicating that the rosettes contain fluorescent material. C) Representative OCT slice of a Nrl knockout mouse revealing details of the nature of the rosettes (arrow) and their depth localization. D) Comparison of the OCT representation of Nrl rosettes with histology (different individual animal). E, F) Detail illustrating how well retinal structures in OCT and histology correlate.

Figure 5. Capability of the OCT to detect and capture the nature of lesions.

A)–D) Site of neovascularization in a Crumbs 1 (Crb1) knockout mouse. A) SLO fluorescence angiographic (FLA) image of a retinal neovascular site (arrow) in a representative Crb1 knockout mouse. B) Detail of g) illustrating the traction the aberrant vessel applies to the neighboring capillaries. C) Representative OCT slice depicting enlarged aberrant retinal vessels (black arrow) as well as choroidal vascular changes (white arrow) at the same position, implicating a connection between both vascular beds. D) Histological section of the above neovascular site for comparison. The black arrow points to aberrant retinal vessels, and the white arrow to choroidal changes.

Figure 6. Topographic analysis of retinal thickness in an organ-specific model of retinoblastoma protein (Rb) deficiency.

Thickness variations (center vs. periphery) were caused by imperfections of the Cre-lox system (see text), leading to differences in developmental apoptosis. A) Histological section across the central retina showing the smooth transition between centrally normal and peripherally reduced thickness. B) OCT section of the same region, the retinal thickness correlating well with the histomorphological data. C) Assessment of the gradual changes of retinal thickness from center to (mid)periphery based on 5 manually placed OCT slices. Left: SLO image of the fundus region with the position of the slices superimposed. Right: OCT slices at the positions indicated, ordered from center to periphery. D) Topography of retinal thickness calculated from 92 equidistant OCT slices (“volume scan” data). The color scale values are in µm.