Three-dimensional optical coherence tomography imaging of retinal sheet implants in live rats

https://doi.org/10.1016/j.jneumeth.2010.02.018Get rights and content

Abstract

Purpose

To obtain three-dimensional images from retinal transplants in live animals and evaluate the placement and structural quality of the transplants.

Methods

Donor retinal sheets were isolated from E19 fetuses of transgenic rats expressing human alkaline phosphatase (hPAP), and transplanted to the subretinal space of 19–56 days old S334ter-3 rat recipients with fast retinal degeneration (average age at surgery 32 days). A total of 143 rats were imaged 1 day to 2.8 months after surgery, using a Fourier-domain optical coherence tomography (FDOCT) system, with an axial resolution of 3.5 μm. The CCD A-line integration time was set at 200 μs for better visualization of degenerated retina. After targeting the transplant area, 139 or 199 consecutive slices were scanned. Projection images and movies of the retinal transplant area were computed and later compared with histology.

Results

OCT scans identified 137 of 141 transplants as a thickening of the degenerated retina. OCT indicated the laminar structure of the transplants and surgical defects, such as RPE/choroid damage with an accuracy rate between 83 and 99%. Three-dimensional projections showed the transplant position in the retina in relation to the optic disc. Histology of transplants by hPAP and hematoxylin–eosin staining was correlated with the OCT results.

Conclusions

Optical coherence tomography is an excellent tool to image retinal layers in a live rat. This procedure helps to evaluate the placement and quality of the transplants in the living eye.

Introduction

Transplantation of retinal sheets (review: Aramant and Seiler, 2004, Seiler and Aramant, 2005) aims at replacing photoreceptors and/or retinal pigment epithelium (RPE) (and other retinal cells) lost in retinal diseases such as age-related macular degeneration (AMD) or retinitis pigmentosa (RP). AMD is the leading cause of blindness among the elderly, affecting about 8 million patients in the U.S. alone (Ding et al., 2009, Jager et al., 2008), and retinitis pigmentosa is an inherited disease that affects about a million patients worldwide (Kennan et al., 2005). The inner retina remains still functional for the some time after photoreceptor loss (Humayun et al., 1999), so it may be possible to restore visual function if newly replaced retinal cells can connect with the remaining host circuitry. Using a special implantation instrument, it has been possible to gently transplant sheets of fetal retina to the subretinal space in rodent retinal degeneration models (Aramant and Seiler, 2004, Seiler and Aramant, 2005) and in human patients (Radtke et al., 2008).

The surgery in the small rat eye is however very challenging since the surgeon cannot observe where the tissue is placed. Only about 20–30% of all transplants develop a normal lamination with photoreceptor outer segments facing the host RPE, in contrast to balls of photoreceptors in rosettes (Seiler and Aramant, 1998). It is very difficult to judge the quality of transplants in fundus exams due to the transparency of the transplant and host retina. To save resources, it would be important to eliminate transplanted rats with surgical defects early on from the study. Therefore, we developed a systematic approach to evaluate transplants in live rats by three-dimensional ocular coherence tomography (OCT) which provides a means to analyze structures in the living eye (Jeon et al., 2008b). In contrast to a previous study (Thomas et al., 2006) that used a commercially available time domain Zeiss Stratus OCT, the OCT setup for this study used serial scans of a Fourier-domain OCT to provide a three-dimensional image of the transplant in the eye (Jeon et al., 2008a, Leitgeb et al., 2003, Zhang et al., 2004). The purpose of this study was to evaluate the accuracy of the 3D FDOCT to predict later histological results.

Section snippets

Animals

For all experimental procedures, animals were treated in accordance with the NIH guidelines for the care and use of laboratory animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, under a protocol approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. Pigmented S334ter-line-3 rats (Liu et al., 1999, Sagdullaev et al., 2003, Seiler et al., 2008a) with fast retinal degeneration were used in this study. The rats were

Results

An overview of the results is shown in Table 1, Table 2, Table 3. OCT imaging identified most of the large transplants, but some small transplants were missed, or the transplant appeared to be smaller than the later histology results showed. Reverse mistakes were more rare (Table 1). Regarding lamination, the overall accuracy rate to identify laminated areas in the transplants were 87%. In 9% of the experiments, a laminated area was identified in OCT scans, which was later not found in sections

Discussion

Fourier-domain 3D OCT has become more of a routine in the clinical setting (Cense et al., 2006, Chen et al., 2009, Rao et al., 2008, Srinivasan et al., 2008, Srinivasan et al., 2006b), but imaging the small rat eye is more of a challenge. The first study to image a rodent eye, published in 2001 (Li et al., 2001), could show differences in retinal thickness, but not much else. Two studies published in 2006 and 2007 showed a high resolution of retinal layers in rodents by spectral domain OCT (

Acknowledgements

This work was supported by the Lincy Foundation, National Institutes of Health (EB-00293, NCI-91717, RR-01192), and the Air Force Office of Science Research (FA9550-04-1-0101). Institutional support from the Beckman Laser Institute and Medical Clinic is also gratefully acknowledged. The authors thank Lakshmi Patil for technical assistance.

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    1

    Both authors contributed equally to the manuscript.

    2

    Current address: Department of Biomedical Engineering, Washington University, St. Louis, MO, United States.

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