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1.  Optical Coherence Tomography for Age-Related Macular Degeneration and Diabetic Macular Edema 
Executive Summary
The purpose of this evidence-based review was to examine the effectiveness and cost-effectiveness of spectral-domain (SD) optical coherence tomography (OCT) in the diagnosis and monitoring of patients with retinal disease, specifically age-related macular degeneration (AMD) and diabetic macular edema (DME). Specifically, the research question addressed was:
What is the sensitivity and specificity of spectral domain OCT relative to the gold standard?
Clinical Need: Target Population and Condition
The incidence of blindness has been increasing worldwide. In Canada, vision loss in those 65 years of age and older is primarily due to AMD, while loss of vision in those 18 years of age and older is mainly due to DME. Both of these conditions are diseases of the retina, which is located at the back of the eye. At the center of the retina is the macula, a 5 mm region that is responsible for what we see in front of us, our ability to detect colour, and fine detail. Damage to the macula gives rise to vision loss, but early detection of asymptomatic disease may lead to the prevention or slowing of the vision loss process.
There are two main types of AMD, ‘dry’ and ‘wet’. Dry AMD is the more prevalent of the two, accounting for approximately 85% of cases and characterized by small deposits of extracellular material called “drusen” that build up in Bruch’s membrane of the eye. Central vision loss is gradual with blurring and eventual colour fading. Wet AMD is a less prevalent condition (15% of all AMD cases) but it accounts for 90% of severe cases. It’s characterized by the appearance of retinal fluid with vision loss due to abnormal blood vessels/leakage within weeks to months of diagnosis. In 2003, the Canadian National Institute for the Blind (CNIB) prevalence estimate for AMD was 1 million Canadians, including approximately 400,000 affected Ontarians. The incidence in 2003 was estimated to be 78,000 new cases in Canada, with approximately one-third of these cases arising in Ontario (n=26,000). Over the next 25 years, the number of new cases is expected to triple.
DME is caused by complications of diabetes mellitus, both Type 1 and Type 2. It is estimated that 1-in-4 persons with diabetes has this condition, though it occurs more frequently among those with type 2 diabetes. The condition is characterized by a swelling of the retina caused by leakage of blood vessels at the back of the eye. In early stages of the disease, vision may still be normal but it can degrade rapidly in later stages. In 2003, the CNIB prevalence estimate for DME was 0.5 million Canadians, with approximately 200,000 Ontarians affected. The incidence of DME is more difficult to ascertain; however, based on an annual incidence rate of 0.8% (for those 20 years of age or older) and the assumption that 1-in-4 persons with diabetes is affected, the incidence of DME in Ontario is estimated to be 21,000 new cases per year.
Optical Coherence Tomography
Prior to the availability of OCT, the standard of care in the diagnosis and/or monitoring of retinal disease was serial testing with fluorescein angiography (FA), biomicroscopy (BM), and stereo-fundus photography (SFP). Each of these is a qualitative measure of disease based on subjective evaluations that are largely dependent on physician expertise. OCT is the first quantitative visual test available for the diagnosis of eye disease. As such, it is allows for a more objective evaluation of the presence/absence of retinal disease and it is the only test that provides a measure of retinal thickness. The technology was developed at the Michigan Institute of Technology (MIT) in 1991 as a real-time imaging modality and is considered comparable to histology. It’s a light-wave based technology producing cross-sectional images with scan rates and resolution parameters that have greatly improved over the last 10 years. It’s also a non-invasive, non-contact visual test that requires just 3 to 5 minutes to assess both eyes.
There are two main types of OCT system, both licensed by Health Canada as class II devices. The original patent was based on a time domain (TD) system (available from 1995) that had an image rate of 100 to 400 scans per second and provided information for a limited view of the retina with a resolution in the range of 10 to 20 μm. The newer system, spectral domain (SD) OCT, has been available since 2006. Improvements with this system include (i) a faster scan speed of approximately 27,000 scans per second; (ii) the ability to scan larger areas of the retina by taking six scans radially-oriented 30 degrees from each other; (iii) increased resolution at 5μm; and (iv) ‘real-time registration,’ which was not previously available with TD.
The increased scan speed of SD systems enables the collection of additional real-time information on larger regions of the retina, thus, reducing the reliance on assumptions required for retinal thickness and volume estimates based on software algorithms. The faster scan speed also eliminates image distortion arising from patient movement (not previously possible with TD), while the improvement in resolution allows for clearer and more distinguishable retinal layers with the possibility of detecting earlier signs of disease. Real-time registration is a new feature of SD that enables the identification of specific anatomical locations on the retina, against which subsequent tests can be evaluated. This is of particular importance in the monitoring of patients. In the evaluation of treatment effects, for example, this enables the same anatomic retinal location to be identified at each visit.
Literature Search
A literature search was performed on February 13, 2009 using Ovid MEDLINE, MEDLINE In-Process & Other Non-Indexed Citations, EMBASE, the Cumulative Index to Nursing & Allied Health Literature (CINAHL), the Cochrane Library, and the International Agency for Health Technology Assessment (INAHTA) for studies published from January 2003 to February 2009. The subject headings and keywords searched included AMD, DME, and OCT (the detailed search strategy can be viewed in Appendix 1). Excluded were case reports, comments, editorials, non-systematic reviews, and letters. Abstacts were reviewed by a single reviewer and, for those studies meeting the eligibility criteria, full-text articles were obtained. In total, 542 articles were included for review.
English-language articles and health technology assessments.
RCTs and observational studies of OCT and AMD or DME.
Studies focusing on either diagnosis or monitoring of disease.
Studies in which outcomes were not specific to those of interest in this report.
Studies of pediatric populations.
Studies on OCT as a screening tool.
Studies that did not assess comparative effectiveness of OCT with a referent, as specified below in “Comparisons of Interest”.
Outcomes of Interest
Studies of sensitivity, specificity.
Comparisons of Interest
Evidence exists for the following comparisons of interest:
OCT compared with the reference “fluorescein angiography” for AMD.
OCT compared with the reference “biomicroscopy” or “stereo or fundus photography” for DME.
Summary of Existing Evidence
No evidence for the accuracy of SD OCT compared to either FA, BM or SFP was published between January 2006 to February 2009; however, two technology assessments were found, one from Alberta and the other from Germany, both of which contain evidence for TD OCT. Although these HTAs included eight studies each, only one study from each report was specific to this review. Additionally, one systematic review was identified for OCT and DME. It is these three articles, all pertaining to time and not spectral domain OCT, as well as comments from experts in the field of OCT and retinal disease, that comprise the evidence contained in this review.
Upon further assessment and consultations with experts in the methodology of clinical test evaluation, it was concluded that these comparators could not be used as references in the evaluation of OCT. The main conclusion was that, without a third test as an arbiter, it is not possible to directly compare the sensitivity and specificity of OCT relative to either FA for AMD and stereo- or fundus – photography for DME. Therefore, in the absence of published evidence, it was deemed appropriate to consult a panel of experts for their views and opinions on the validity of OCT and its utility in clinical settings. This panel consisted of four clinicians with expertise in AMD and/or DME and OCT, as well as a medical biophysicist with scientific expertise in ocular technologies. This is considered level 5 evidence, but in the absence of an appropriate comparator for further evaluation of OCT, this may be the highest level of evidence possible.
Summary of Findings
The conclusions for SD OCT based on Level 5 evidence, or expert consultation, are as follows:
OCT is considered an essential part of the diagnosis and follow-up of patients with DME and AMD.
OCT is adjunctive to FA for both AMD and DME but should decrease utilization of FA as a monitoring modality.
OCT will result in a decline in the use of BM in the monitoring of patients with DME, given its increased accuracy and consistency.
OCT is diffusing rapidly and the technology is changing. Since FA is still considered pivotal in the diagnosis and treatment of AMD and DME, and there is no common outcome against which to compare these technologies, it is unlikely that RCT evidence of efficacy for OCT will ever be forthcoming.
In addition to the accuracy of OCT in the detection of disease, assessment of the clinical utility of this technology included a rapid review of treatment effects for AMD and DME. The treatment of choice for AMD is Lucentis®, with or without Avastin® and photodynamic therapy. For DME the treatment of choice is laser photocoagulation, which may be replaced with Lucentis® injections (Expert consultation). The evidence, as presented in systematic reviews and other health technology assessments, indicates that there are effective treatments available for both AMD and DME.
Considerations for the Ontario Health System
OCT testing is presently an uninsured service in Ontario with patients paying approximately $150 out-of-pocket per test. Several provinces do provide funding for this procedure, including British Columbia, Alberta, Saskatchewan, Newfoundland, Nova Scotia, Prince Edward Island, and the Yukon Territory. Provinces that do not provide such funding are Quebec, Manitoba and New Brunswick.
The demand for OCT is expected to increase with aging of the population.
PMCID: PMC3377511  PMID: 23074517
2.  Human CRB1-Associated Retinal Degeneration: Comparison with the rd8 Crb1-Mutant Mouse Model 
The common form of Leber congenital amaurosis (LCA) due to CRB1 mutations needs further characterization of the human disease and a test of relevance of currently available animal models. A cohort of these patients was evaluated, and the disease expression was compared to that of the rd8 mouse model, to seek answers to questions of how to move CRB1-LCA closer to therapy.
To investigate the human disease due to CRB1 mutations and compare results with the Crb1-mutant rd8 mouse.
Twenty-two patients with CRB1 mutations were studied. Function was assessed with perimetry and electroretinography (ERG) and retinal structure with optical coherence tomography (OCT). Cortical structure and function were quantified with magnetic resonance imaging (MRI). Rd8 mice underwent ERG, OCT, and retinal histopathology.
Visual acuities ranged from 20/25 to light perception. Rod ERGs were not detectable; small cone signals were recordable. By perimetry, small central visual islands were separated by midperipheral scotomas from far temporal peripheral islands. The central islands were cone mediated, whereas the peripheral islands retained some rod function. With OCT, there were small foveal islands of thinned outer nuclear layer (ONL) surrounded by thick delaminated retina with intraretinal hyperreflective lesions. MRI showed structurally normal optic nerves and only subtle changes to occipital lobe white and gray matter. Functional MRI indicated that whole-brain responses from patients were of reduced amplitude and spatial extent compared with those of normal controls. Rd8 mice had essentially normal ERGs; OCT and histopathology showed patchy retinal disorganization with pseudorosettes more pronounced in ventral than in dorsal retina. Photoreceptor degeneration was associated with dysplastic regions.
CRB1 mutations lead to early-onset severe loss of vision with thickened, disorganized, nonseeing retina. Impaired peripheral vision can persist in late disease stages. Rd8 mice also have a disorganized retina, but there is sufficient photoreceptor integrity to produce largely normal retinal function. Differences between human and mouse diseases will complicate proof-of-concept studies intended to advance treatment initiatives.
PMCID: PMC3176016  PMID: 21757580
3.  Comparison of Ultrahigh- and Standard-Resolution Optical Coherence Tomography for Imaging Macular Pathology 
Ophthalmology  2005;112(11):1922.e1-1922.15.
To compare ultrahigh-resolution optical coherence tomography (UHR OCT) with standard-resolution OCT for imaging macular diseases, develop baselines for interpreting OCT images, and identify situations where UHR OCT can provide additional information on disease morphology.
Cross-sectional study.
One thousand two eyes of 555 patients with different macular diseases including macular hole, macular edema, central serous chorioretinopathy, age-related macular degeneration (AMD), choroidal neovascularization, epiretinal membrane, retinal pigment epithelium (RPE) detachment, and retinitis pigmentosa.
A UHR ophthalmic OCT system that achieves 3-μm axial image resolution was developed for imaging in the ophthalmology clinic. Comparative studies were performed with both UHR OCT and standard 10-μm-resolution OCT. Standard scanning protocols of 6 radial 6-mm scans through the fovea were obtained with both systems. Ultrahigh-resolution OCT and standard-resolution OCT images were correlated with standard ophthalmic examination techniques (dilated ophthalmoscopy, fluorescein angiography, indocyanine green angiograms) to assess morphological information contained in the images.
Main Outcome Measures
Ultrahigh-resolution and standard-resolution OCT images of macular pathologies.
Correlations of UHR OCT images, standard-resolution images, fundus examination, and/or fluorescein angiography were demonstrated in full-thickness macular hole, central serous chorioretinopathy, macular edema, AMD, RPE detachment, epiretinal membrane, vitreal macular traction, and retinitis pigmentosa. Ultrahigh-resolution OCT and standard-resolution OCT exhibited comparable performance in differentiating thicker retinal layers, such as the retinal nerve fiber, inner and outer plexiform, and inner and outer nuclear. Ultrahigh-resolution OCT had improved performance differentiating finer structures or structures with lower contrast, such as the ganglion cell layer and external limiting membrane. Ultrahigh-resolution OCT confirmed the interpretation of features, such as the boundary between the photoreceptor inner and outer segments, which is also visible in standard-resolution OCT. The improved resolution of UHR OCT is especially advantageous in assessing photoreceptor morphology.
Ultrahigh-resolution OCT enhances the visualization of intraretinal architectural morphology relative to standard-resolution OCT. Ultrahigh-resolution OCT images can provide a baseline for defining the interpretation of standard-resolution images, thus enhancing the clinical utility of standard OCT imaging. In addition, UHR OCT can provide additional information on macular disease morphology that promises to improve understanding of disease progression and management.
PMCID: PMC1937402  PMID: 16183127
4.  In vivo imaging of the Mouse Model of X-Linked Juvenile Retinoschisis Using Fourier Domain Optical Coherence Tomography 
The purpose of this study is to investigate Fourier Domain Optical Coherence Tomography (FD OCT) as a non-invasive tool for retinal imaging in the Rs1h knockout mouse (model for X-linked Juvenile Retinoschisis).
A prototype spectrometer based FD OCT system was used in combination with a custom optical beam-scanning platform. Images of the retinas from wild type and Rs1h knockout mice were acquired non-invasively using FD OCT with the specimen anesthetized. At the completion of the non-invasive FD OCT imaging, invasive retinal cross sectional images (histology) were acquired from a nearby region for comparison to the FD OCT images.
The retinal layers could be identified in the FD OCT images, permitting delineation and thickness measurement of the outer nuclear layer (ONL). During FD OCT in vivo imaging of the Rs1h knockout mouse, holes were observed in the inner nuclear layer (INL) and retinal cell disorganization was observed as a change in the backscattering intensity profile. Comparison of the ONL measurements acquired non-invasively using FD OCT to measurements taken using histology at nearby locations showed a degeneration of roughly thirty percent of the ONL by the age of two months in Rs1h knockout mice relative to wild type.
FD OCT has been demonstrated for non-invasive imaging of retinal degeneration and observation of retinal holes in Rs1h knockout mice.
PMCID: PMC2693243  PMID: 19182246
5.  Spectral-Domain Optical Coherence Tomography of the Rodent Eye: Highlighting Layers of the Outer Retina Using Signal Averaging and Comparison with Histology 
PLoS ONE  2014;9(5):e96494.
Spectral-Domain Optical Coherence Tomography (SD-OCT) is a widely used method to observe retinal layers and follow pathological events in human. Recently, this technique has been adapted for animal imaging. This non-invasive technology brings a cross-sectional visualization of the retina, which permits to observe precisely each layer. There is a clear expansion of the use of this imaging modality in rodents, thus, a precise characterization of the different outer retinal layers observed by SD-OCT is now necessary to make the most of this technology. The identification of the inner strata until the outer nuclear layer has already been clearly established, while the attribution of the layers observed by SD-OCT to the structures corresponding to photoreceptors segments and retinal pigment epithelium is much more questionable. To progress in the understanding of experimental SD-OCT imaging, we developed a method for averaging SD-OCT data to generate a mean image allowing to better delineate layers in the retina of pigmented and albino strains of mice and rats. It allowed us to locate precisely the interface between photoreceptors and retinal pigment epithelium and to identify unambiguously four layers corresponding to the inner and outer parts of photoreceptors segments. We show that the thickness of the various layers can be measured as accurately in vivo on SD-OCT images, than post-mortem by a morphometric analysis of histological sections. We applied SD-OCT to different models and demonstrated that it allows analysis of focal or diffuse retinal pathological processes such as mutation-dependant damages or light-driven modification of photoreceptors. Moreover, we report a new method of combined use of SD-OCT and integration to quantify laser-induced choroidal neovascularization. In conclusion, we clearly demonstrated that SD-OCT represents a valuable tool for imaging the rodent retina that is at least as accurate as histology, non-invasive and allows longitudinal follow-up of the same animal.
PMCID: PMC4008571  PMID: 24788712
6.  Spectral Domain Optical Coherence Tomography in Mouse Models of Retinal Degeneration 
Spectral domain optical coherence tomography (SD-OCT) allows cross-sectional visualization of retinal structures in vivo. Here, we report the efficacy of a commercially available SD-OCT device to study mouse models of retinal degeneration.
C57BL/6 and BALB/c wild type mice and three different mouse models of hereditary retinal degeneration (Rho-/-, rd1, RPE65-/-) were investigated using confocal scanning laser ophthalmoscopy (cSLO) for en face visualization and SD-OCT for cross-sectional imaging of retinal structures. Histology was performed to correlate structural findings in SD-OCT with light microscopic data.
In C57BL/6 and BALB/c mice, cSLO and SD-OCT imaging provided structural details of frequently used control animals (central retinal thickness, CRTC57BL/6 = 237±2μm and CRTBALB/c = 211±10μm). RPE65-/- mice at 11 months of age showed a significant reduction of retinal thickness (CRTRPE65 = 193±2μm) with thinning of the outer nuclear layer. Rho-/- mice at P28 demonstrated degenerative changes mainly in the outer retinal layers (CRTRho = 193±2μm). Examining rd1 animals before and after the onset of retinal degeneration allowed to monitor disease progression (CRTrd1 P11 = 246±4μm, CRTrd1 P28 = 143±4μm). Correlation of CRT assessed by histology and SD-OCT was high (r2 = 0.897).
We demonstrated cross sectional visualization of retinal structures in wild type mice and mouse models for retinal degeneration in vivo using a commercially available SD-OCT device. This method will help to reduce numbers of animals needed per study by allowing longitudinal study designs and facilitate characterization of disease dynamics and evaluation of putative therapeutic effects following experimental interventions.
PMCID: PMC2800101  PMID: 19661229
optical coherence tomography; retinal degeneration; imaging; mouse models
7.  Non-invasive assessment of retinal alterations in mouse models of infantile and juvenile neuronal ceroid lipofuscinosis by spectral domain optical coherence tomography 
The neuronal ceroid lipofuscinoses constitute a group of fatal inherited lysosomal storage diseases that manifest in profound neurodegeneration in the CNS. Visual impairment usually is an early symptom and selective degeneration of retinal neurons has been described in patients suffering from distinct disease subtypes. We have previously demonstrated that palmitoyl protein thioesterase 1 deficient (Ppt1-/-) mice, a model of the infantile disease subtype, exhibit progressive axonal degeneration in the optic nerve and loss of retinal ganglion cells, faithfully reflecting disease severity in the CNS. Here we performed spectral domain optical coherence tomography (OCT) in Ppt1-/- and ceroid lipofuscinosis neuronal 3 deficient (Cln3-/-) mice, which are models of infantile and juvenile neuronal ceroid lipofuscinosis, respectively, in order to establish a non-invasive method to assess retinal alterations and monitor disease severity in vivo.
Blue laser autofluorescence imaging revealed increased accumulation of autofluorescent storage material in the inner retinae of 7-month-old Ppt1-/- and of 16-month-old Cln3-/- mice in comparison with age-matched control littermates. Additionally, optical coherence tomography demonstrated reduced thickness of retinae in knockout mice in comparison with age-matched control littermates. High resolution scans and manual measurements allowed for separation of different retinal composite layers and revealed a thinning of layers in the inner retinae of both mouse models at distinct ages. OCT measurements correlated well with subsequent histological analysis of the same retinae.
These results demonstrate the feasibility of OCT to assess neurodegenerative disease severity in mouse models of neuronal ceroid lipofuscinosis and might have important implications for diagnostic evaluation of disease progression and therapeutic efficacy in patients. Moreover, the non-invasive method allows for longitudinal studies in experimental models, reducing the number of animals used for research.
PMCID: PMC4035096  PMID: 24887158
Optical coherence tomography; Neuronal ceroid lipofuscinosis; Neurodegeneration; Retinal degeneration; Lysosomal storage disease
8.  Long-Term Characterization of Retinal Degeneration in rd1 and rd10 Mice Using Spectral Domain Optical Coherence Tomography 
We characterize the in vivo changes over time in the retinal structure of wild-type mice alongside two lines of mice deficient in the β-subunit of phosphodiesterase (rd1 and rd10 mice) using spectral domain optical coherence tomography (SD-OCT).
SD-OCT images were obtained using the Bioptigen spectral domain ophthalmic imaging system (SDOIS). Wild-type C57BL/6J, rd1 and rd10 mice ranging in age from P14 to P206 were sedated with 1% isoflurane. Horizontal and vertical linear scans through the optic nerve, and annular scans around the optic nerve were obtained.
SD-OCT imaging of wild-type mice demonstrated visibility of the inner segment/outer segment (IS/OS) junction, external limiting membrane (ELM), outer nuclear layer (ONL), and outer plexiform layer (OPL). At P14, most rd10 mice exhibited normal SD-OCT profiles, but some displayed changes in the IS/OS junction. At the same time point, rd1 mice had severe outer retinal degeneration. In rd10 mice, imaging revealed loss of the IS/OS junction by P18, hyperreflective changes in the ONL at P20, hyperreflective vitreous opacities, and shallow separation of the neural retina from the RPE. Retinal separations were not observed in rd1 mice. Segmentation analysis in wild-type mice demonstrated relatively little variability between animals, while in rd10 and rd1 mice there was a steady decline in outer retinal thickness. Histologic studies demonstrated correlation of retinal features with those seen on SD-OCT scans. Segmentation analysis provides a quantitative and reproducible method for measuring in vivo retinal changes in mice.
SD-OCT provides a non-invasive method of following long-term retinal changes in mice in vivo. Although rd10 and rd1 mice have mutations in the same gene, they demonstrate significantly different features on SD-OCT.
SD-OCT imaging in rd1 and rd10 mice provided quantitative measurements of inner and outer retinal thickness and revealed several previously unreported results including frequent in vivo separation of the retina from the retinal pigment epithelium in the rd10 mouse, but not in the rd1 mouse.
PMCID: PMC3394742  PMID: 22562504
9.  Analysis of Peripapillary Atrophy using Spectral Domain Optical Coherence Tomography 
Ophthalmology  2011;118(3):531-536.
To study retinal morphological changes around the optic disc in patients with peripapillary atrophy (PPA) with high-resolution spectral domain optical coherence tomography (SD OCT).
Cross-sectional, retrospective analysis
One hundred and three eyes of 73 patients with PPA and 21 eyes of 12 normal patients seen at the New England Eye Center, Tufts Medical Center between January 2007 and August 2009.
SD OCT images taken through the region of PPA were quantitatively and qualitatively analyzed. Inclusion criteria included eyes with at least 300 μm of temporal PPA as detected on color fundus photographs. The study population was divided into subgroups according to the following clinical diagnoses: glaucoma (n=13), age-related macular degeneration (n=11), high myopia (n=11), glaucoma and high myopia (n=3), and optic neuropathy (n=11). Fifty-four patients were classified with other diagnoses. Using OCT software, retinal thickness and retinal nerve fiber layer thickness (RNFL) were both manually measured perpendicular to the internal limiting membrane and retinal pigment epithelium (RPE) 300 μm temporal to the optic disc, within the region of peripapillary atrophy. Qualitative analysis for morphological changes in the atrophic area was also performed.
Main outcome measures
Qualitative assessment and quantitative measures of retinal and retinal nerve fiber layer thickness in PPA.
The study group was categorized by 6 characteristics demonstrated in the area of PPA by SD OCT: RPE loss with accompanying photoreceptor loss, RPE disruption, RNFL thickening with plaque-like formation, intraretinal cystic changes, inner and outer retinal thinning, and abnormal retinal sloping. Statistical analysis of measurements revealed a statistically significant difference in the total retinal thickness between normal eyes and eyes with PPA (p = 0.0005), with normals 15% thicker than the PPA group; however, the RNFL thickness was not significantly different between the normal and PPA group (p = 0.05).
Eyes with peripapillary atrophy manifest characteristic retinal changes that can be described via SD OCT.
PMCID: PMC3017221  PMID: 20920826
10.  Optical Coherence Tomography of Retinal Degeneration in Royal College of Surgeons Rats and Its Correlation with Morphology and Electroretinography 
PLoS ONE  2016;11(9):e0162835.
To evaluate the correlation between optical coherence tomography (OCT) and the histological, ultrastructural and electroretinography (ERG) findings of retinal degeneration in Royal College of Surgeons (RCS-/-) rats.
Materials and Methods
Using OCT, we qualitatively and quantitatively observed the continual retinal degeneration in RCS-/- rats, from postnatal (PN) day 17 until PN day 111. These findings were compared with the corresponding histological, electron microscopic, and ERG findings. We also compared them to OCT findings in wild type RCS+/+ rats, which were used as controls.
After PN day 17, the hyperreflective band at the apical side of the photoreceptor layer became blurred. The inner segment (IS) ellipsoid zone then became obscured, and the photoreceptor IS and outer segment (OS) layers became diffusely hyperreflective after PN day 21. These changes correlated with histological and electron microscopic findings showing extracellular lamellar material that accumulated in the photoreceptor OS layer. After PN day 26, the outer nuclear layer became significantly thinner (P < 0.01) and hyperreflective compared with that in the controls; conversely, the photoreceptor IS and OS layers, as well as the inner retinal layers, became significantly thicker (P < 0.001 and P = 0.05, respectively). The apical hyperreflective band, as well as the IS ellipsoid zone, gradually disappeared between PN day 20 and PN day 30; concurrently, the ERG a- and b-wave amplitudes deteriorated. In contrast, the thicknesses of the combined retinal pigment epithelium and choroid did not differ significantly between RCS-/- and RCS+/+ rats.
Our results suggest that OCT demonstrates histologically validated photoreceptor degeneration in RCS rats, and that OCT findings partly correlate with ERG findings. We propose that OCT is a less invasive and useful method for evaluating photoreceptor degeneration in animal models of retinitis pigmentosa.
PMCID: PMC5028068  PMID: 27644042
11.  Three-dimensional Retinal Imaging with High-Speed Ultrahigh-Resolution Optical Coherence Tomography 
Ophthalmology  2005;112(10):1734-1746.
To demonstrate high-speed, ultrahigh-resolution, 3-dimensional optical coherence tomography (3D OCT) and new protocols for retinal imaging.
Ultrahigh-resolution OCT using broadband light sources achieves axial image resolutions of ~2 μm compared with standard 10-μm-resolution OCT current commercial instruments. High-speed OCT using spectral/Fourier domain detection enables dramatic increases in imaging speeds. Three-dimensional OCT retinal imaging is performed in normal human subjects using high-speed ultrahigh-resolution OCT. Three-dimensional OCT data of the macula and optic disc are acquired using a dense raster scan pattern. New processing and display methods for generating virtual OCT fundus images; cross-sectional OCT images with arbitrary orientations; quantitative maps of retinal, nerve fiber layer, and other intraretinal layer thicknesses; and optic nerve head topographic parameters are demonstrated.
Three-dimensional OCT imaging enables new imaging protocols that improve visualization and mapping of retinal microstructure. An OCT fundus image can be generated directly from the 3D OCT data, which enables precise and repeatable registration of cross-sectional OCT images and thickness maps with fundus features. Optical coherence tomography images with arbitrary orientations, such as circumpapillary scans, can be generated from 3D OCT data. Mapping of total retinal thickness and thicknesses of the nerve fiber layer, photoreceptor layer, and other intraretinal layers is demonstrated. Measurement of optic nerve head topography and disc parameters is also possible. Three-dimensional OCT enables measurements that are similar to those of standard instruments, including the StratusOCT, GDx, HRT, and RTA.
Three-dimensional OCT imaging can be performed using high-speed ultrahigh-resolution OCT. Three-dimensional OCT provides comprehensive visualization and mapping of retinal microstructures. The high data acquisition speeds enable high-density data sets with large numbers of transverse positions on the retina, which reduces the possibility of missing focal pathologies. In addition to providing image information such as OCT cross-sectional images, OCT fundus images, and 3D rendering, quantitative measurement and mapping of intraretinal layer thickness and topographic features of the optic disc are possible. We hope that 3D OCT imaging may help to elucidate the structural changes associated with retinal disease as well as improve early diagnosis and monitoring of disease progression and response to treatment.
PMCID: PMC1939719  PMID: 16140383
12.  Outer Retinal Tubulation in Advanced Age-Related Macular Degeneration: Optical Coherence Tomographic Findings Correspond to Histology 
Retina (Philadelphia, Pa.)  2015;35(7):1339-1350.
To compare optical coherence tomography (OCT) and histology of outer retinal tubulation (ORT) secondary to advanced age-related macular degeneration (AMD) in patients and in post-mortem specimens, with particular attention to the basis of the hyper-reflective border of ORT.
A private referral practice (imaging) and an academic research laboratory (histology) collaborated on two retrospective case series. High-resolution OCT raster scans of 43 eyes (34 patients) manifesting ORT secondary to advanced AMD were compared to high-resolution histological sections through the fovea and superior perifovea of donor eyes (13 atrophic AMD and 40 neovascular AMD) preserved ≤4 hours after death.
ORT seen on OCT corresponded to histologic findings of tubular structures comprised largely of cones lacking outer segments (OS) and lacking inner segments (IS). Four phases of cone degeneration were histologically distinguishable in ORT lumenal walls, nascent, mature, degenerate, and end-stage (IS and OS; IS only; no IS; no photoreceptors and only Müller cells forming external limiting membrane, ELM, respectively). Mitochondria, which are normally long and bundled within IS ellipsoids, were small and scattered within shrunken IS and cell bodies of surviving cones. A lumenal border was delimited by an ELM. ORT observed in closed and open configurations were distinguishable from cysts and photoreceptor islands on both OCT and histology. Hyper-reflective lumenal material seen on OCT represents trapped retinal pigment epithelium (RPE) and non-RPE cells.
The defining OCT features of ORT are location in the outer nuclear layer (ONL), a hyper-reflective band differentiating it from cysts, and RPE that is either dysmorphic or absent. ORT histologic and OCT findings corresponded in regard to composition, location, shape, and stages of formation. The reflectivity of ORT lumenal walls on OCT apparently does not require an OS or an IS/OS junction, indicating an independent reflectivity source, possibly mitochondria, in the IS.
PMCID: PMC4478232  PMID: 25635579
Photoreceptors; Müller cells; age-related macular degeneration; outer retinal tubulation; ellipsoid; reflectivity; optical coherence tomography; histology; transmission electron microscopy; mitochondria
13.  The BALB/c mouse: Effect of standard vivarium lighting on retinal pathology during aging 
Experimental eye research  2015;135:192-205.
BALB/cJ mice housed under normal vivarium lighting conditions can exhibit profound retinal abnormalities, including retinal infoldings, autofluorescent inflammatory cells, and photoreceptor degeneration. To explore the sensitivity of the outer retina to cyclic lighting during aging, a cohort of BALB/cJ mice was evaluated with Scanning Laser Ophthalmoscopy (SLO), Spectral-Domain Optical Coherence Tomography (OCT) and conventional histopathology. Mice were bred and reared in a low-illuminance (extracage/intracage: 13 lx/1 lx) vivarium under cyclic light (14 h light: 10 h dark). Retinal imaging (around postnatal day 70) was performed to screen for any pre-existing abnormalities and to establish a baseline. Mice with normal retinas were separated into groups (A, B, C) and placed on bottom (Groups A & B) or top (Group C) of the cage racks where cage illumination was <10 & 150 lx respectively. Experimental groups B & C were imaged multiple times over a 17 month period. Mice from group A (controls) were imaged only once post-baseline at various times for comparison to groups B & C. Mice were assessed by histology at 8, 15, 20, 36, and 56 weeks and immunohistochemistry at 15 weeks post-baseline. SLO and OCT retinal images were measured and the resulting trends displayed as a function of age and light exposure. Retinal lesions (RL) and autofluorescent foci (AFF) were identified with histology as photoreceptor layer infoldings (IF) and localized microglia/macrophages (MM), respectively. Few RL and AFF were evident at baseline. Retinal infoldings were the earliest changes followed by subjacent punctate autofluorescent MM. The colocalization of IF and MM suggests a causal relationship. The incidence of these pathological features increased in all groups relative to baseline. OCT imaging revealed thinning of the outer nuclear layer (ONL) in all groups at 1 year relative to baseline. ONL thinning followed an exponential rate of change but the decay constant varied depending on intensity of illumination of the groups. Advanced age and top row illuminance conditions resulted in significant photoreceptor cell loss as judged by decreased thickness of the ONL. Photoreceptor loss was preceded by both retinal infoldings and the presence of autofluorescent inflammatory cells in the outer retina, suggesting that these changes are early indicators of light toxicity in the BALB/cJ mouse.
PMCID: PMC4446204  PMID: 25895728
BALB/c; Mice; Imaging; Retina; Degeneration; Infolding; Inflammation; Phototoxicity
14.  High-Definition and 3-dimensional Imaging of Macular Pathologies with High-speed Ultrahigh-Resolution Optical Coherence Tomography 
Ophthalmology  2006;113(11):2054.e1-2054.14.
To assess high-speed ultrahigh-resolution optical coherence tomography (OCT) image resolution, acquisition speed, image quality, and retinal coverage for the visualization of macular pathologies.
Retrospective cross-sectional study.
Five hundred eighty-eight eyes of 327 patients with various macular pathologies.
High-speed ultrahigh-resolution OCT images were obtained in 588 eyes of 327 patients with selected macular diseases. Ultrahigh-resolution OCT using Fourier/spectral domain detection achieves ~3-μm axial image resolutions, acquisition speeds of ~25 000 axial scans per second, and >3 times finer resolution and >50 times higher speed than standard OCT. Three scan protocols were investigated. The first acquires a small number of high-definition images through the fovea. The second acquires a raster series of high-transverse pixel density images. The third acquires 3-dimensional OCT data using a dense raster pattern. Three-dimensional OCT can generate OCT fundus images that enable precise registration of OCT images with the fundus. Using the OCT fundus images, OCT results were correlated with standard ophthalmoscopic examination techniques.
Main Outcome Measures
High-definition macular pathologies.
Macular holes, age-related macular degeneration, epiretinal membranes, diabetic retinopathy, retinal dystrophies, central serous chorioretinopathy, and other pathologies were imaged and correlated with ophthalmic examination, standard OCT, fundus photography, and fluorescein angiography, where applicable. High-speed ultrahigh-resolution OCT generates images of retinal pathologies with improved quality, more comprehensive retinal coverage, and more precise registration than standard OCT. The speed preserves retinal topography, thus enabling the visualization of subtle changes associated with disease. High-definition high-transverse pixel density OCT images improve visualization of photoreceptor and pigment epithelial morphology, as well as thin intraretinal and epiretinal structures. Three-dimensional OCT enables comprehensive retinal coverage, reduces sampling errors, and enables assessment of 3-dimensional pathology.
High-definition 3-dimensional imaging using high-speed ultrahigh-resolution OCT improves image quality, retinal coverage, and registration. This new technology has the potential to become a useful tool for elucidating disease pathogenesis and improving disease diagnosis and management.
PMCID: PMC1939823  PMID: 17074565
15.  Lentiviral Gene Transfer of Rpe65 Rescues Survival and Function of Cones in a Mouse Model of Leber Congenital Amaurosis 
PLoS Medicine  2006;3(10):e347.
RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice.
Methods and Findings
Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease.
By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65−/− mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose.
In theRpe65-/- mouse model of Leber congenital amaurosis, injection of a lentiviral vector expressing the Rpe65 mouse cDNA was able to prevent cone degeneration and restore cone function.
Editors' Summary
Leber congenital amaurosis (LCA) is the name of a group of hereditary diseases that cause blindness in infants and children. Changes in any one of a number of different genes can cause the blindness, which affects vision starting at birth or soon after. The condition was first described by a German doctor, Theodore Leber, in the 19th century, hence the first part of the name; “amaurosis” is another word for blindness. Mutations in one gene called retinal pigment epithelium-specific protein, 65 kDa (RPE65)—so called because it is expressed in the pigment epithelium, a cell layer adjacent to the light-sensitive cells, and is 65 kilodaltons in size—cause about 10% of cases of LCA. The product of this gene is essential for the recycling of a substance called 11-cis-retinal, which is necessary for the light-sensitive rods and cones of the retina to capture light. If the gene is abnormal, the sensitivity of the retina to light is drastically reduced, but it also leads to damage to the light-sensitive cells themselves.
Why Was This Study Done?
Potentially, eyes diseases such as this one could be treated by gene therapy, which works by replacing a defective gene with a normal functional one, usually by putting a copy of the normal gene into a harmless virus and injecting it into the affected tissue—in this case, the eye. The researchers here wanted to see whether expressing wild-type RPE65 using a particular type of gene vector that can carry large pieces of DNA transcript—a lentiviral vector—could prevent degeneration of cone cells and restore cone function in a mouse model of this type of LCA—mice who had had this Rpe65 gene genetically removed.
What Did the Researchers Do and Find?
Injection of the normal gene into the retina of Rpe65-deficient mice led to sustained expression of the protein RPE65 in the retinal pigment epithelium. Electrical recordings of the activity of the eyes in these mice showed that Rpe65 gene transfer restored retinal function to a near-normal level. In addition, Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mice.
What Do These Findings Mean?
These findings suggest that it is theoretically possible to treat this type of blindness by gene therapy. However, because this study was done in mice, many other steps need to be taken before it will be clear whether the treatment could work in humans. These steps include a demonstration that the virus is safe in humans, and experiments to determine what dose of virus would be needed and how long the effects of the treatment would last. Another question is whether it would be necessary (or even possible) to treat affected children during early childhood or when children start losing vision.
Additional Information.
Please access these Web sites via the online version of this summary at
The Foundation for Retinal Research has detailed information on Leber's congenital amaurosis
Contact a Family is a UK organization that aims to put families of children with illnesses in touch with each other
The Foundation for Fighting Blindness funds research into, and provides information about many types of blindness, including Leber's congenital amaurosis
This Web site provides information on gene therapy clinical trials, including those dedicated to cure eye diseases
This foundation provides information on diseases leading to blindness, including Leber's congenital amaurosis
PMCID: PMC1592340  PMID: 17032058
16.  Quantitative Analysis of Mouse Retinal Layers Using Automated Segmentation of Spectral Domain Optical Coherence Tomography Images 
Quantification of retinal layers using automated segmentation of optical coherence tomography (OCT) images allows for longitudinal studies of retinal and neurological disorders in mice. The purpose of this study was to compare the performance of automated retinal layer segmentation algorithms with data from manual segmentation in mice using the Spectralis OCT.
Spectral domain OCT images from 55 mice from three different mouse strains were analyzed in total. The OCT scans from 22 C57Bl/6, 22 BALBc, and 11 C3A.Cg-Pde6b+Prph2Rd2/J mice were automatically segmented using three commercially available automated retinal segmentation algorithms and compared to manual segmentation.
Fully automated segmentation performed well in mice and showed coefficients of variation (CV) of below 5% for the total retinal volume. However, all three automated segmentation algorithms yielded much thicker total retinal thickness values compared to manual segmentation data (P < 0.0001) due to segmentation errors in the basement membrane.
Whereas the automated retinal segmentation algorithms performed well for the inner layers, the retinal pigmentation epithelium (RPE) was delineated within the sclera, leading to consistently thicker measurements of the photoreceptor layer and the total retina.
Translational Relevance
The introduction of spectral domain OCT allows for accurate imaging of the mouse retina. Exact quantification of retinal layer thicknesses in mice is important to study layers of interest under various pathological conditions.
PMCID: PMC4555843  PMID: 26336634
retinal layer segmentation; optical coherence tomography; retina
17.  Multi-Functional OCT Enables Longitudinal Study of Retinal Changes in a VLDLR Knockout Mouse Model 
PLoS ONE  2016;11(10):e0164419.
We present a multi-functional optical coherence tomography (OCT) imaging approach to study retinal changes in the very-low-density-lipoprotein-receptor (VLDLR) knockout mouse model with a threefold contrast. In the retinas of VLDLR knockout mice spontaneous retinal-chorodoidal neovascularizations form, having an appearance similar to choroidal and retinal neovascularizations (CNV and RNV) in neovascular age-related macular degeneration (AMD) or retinal angiomatous proliferation (RAP). For this longitudinal study, the mice were imaged every 4 to 6 weeks starting with an age of 4 weeks and following up to the age of 11 months. Significant retinal changes were identified by the multi-functional imaging approach offering a threefold contrast: reflectivity, polarization sensitivity (PS) and motion contrast based OCT angiography (OCTA). By use of this intrinsic contrast, the long-term development of neovascularizations was studied and associated processes, such as the migration of melanin pigments or retinal-choroidal anastomosis, were assessed in vivo. Furthermore, the in vivo imaging results were validated with histological sections at the endpoint of the experiment. Multi-functional OCT proves as a powerful tool for longitudinal retinal studies in preclinical research of ophthalmic diseases. Intrinsic contrast offered by the functional extensions of OCT might help to describe regulative processes in genetic animal models and potentially deepen the understanding of the pathogenesis of retinal diseases such as wet AMD.
PMCID: PMC5053493  PMID: 27711217
18.  Retinal Thickening and Photoreceptor Loss in HIV Eyes without Retinitis 
PLoS ONE  2015;10(8):e0132996.
To determine the presence of structural changes in HIV retinae (i.e., photoreceptor density and retinal thickness in the macula) compared with age-matched HIV-negative controls.
Cohort of patients with known HIV under CART (combination Antiretroviral Therapy) treatment were examined with a flood-illuminated retinal AO camera to assess the cone photoreceptor mosaic and spectral-domain optical coherence tomography (SD-OCT) to assess retinal layers and retinal thickness.
Twenty-four eyes of 12 patients (n = 6 HIV-positive and 6 HIV-negative) were imaged with the adaptive optics camera. In each of the regions of interest studied (nasal, temporal, superior, inferior), the HIV group had significantly less mean cone photoreceptor density compared with age-matched controls (difference range, 4,308–6,872 cones/mm2). A different subset of forty eyes of 20 patients (n = 10 HIV-positive and 10 HIV-negative) was included in the retinal thickness measurements and retinal layer segmentation with the SD-OCT. We observed significant thickening in HIV positive eyes in the total retinal thickness at the foveal center, and in each of the three horizontal B-scans (through the macular center, superior, and inferior to the fovea). We also noted that the inner retina (combined thickness from ILM through RNFL to GCL layer) was also significantly thickened in all the different locations scanned compared with HIV-negative controls.
Our present study shows that the cone photoreceptor density is significantly reduced in HIV retinae compared with age-matched controls. HIV retinae also have increased macular retinal thickness that may be caused by inner retinal edema secondary to retinovascular disease in HIV. The interaction of photoreceptors with the aging RPE, as well as possible low-grade ocular inflammation causing diffuse inner retinal edema, may be the key to the progressive vision changes in HIV-positive patients without overt retinitis.
PMCID: PMC4526563  PMID: 26244973
19.  Projection OCT fundus imaging for visualising outer retinal pathology in non-exudative age-related macular degeneration 
To demonstrate ultrahigh-resolution, three-dimensional optical coherence tomography (3D-OCT) and projection OCT fundus imaging for enhanced visualisation of outer retinal pathology in non-exudative age-related macular degeneration (AMD).
A high-speed, 3.5 μm resolution OCT prototype instrument was developed for the ophthalmic clinic. Eighty-three patients with non-exudative AMD were imaged. Projection OCT fundus images were generated from 3D-OCT data by selectively summing different retinal depth levels. Results were compared with standard ophthalmic examination, including fundus photography and fluorescein angiography, when indicated.
Projection OCT fundus imaging enhanced the visualisation of outer retinal pathology in non-exudative AMD. Different types of drusen exhibited distinct features in projection OCT images. Photoreceptor disruption was indicated by loss of the photoreceptor inner/outer segment (IS/OS) boundary and external limiting membrane (ELM). RPE atrophy can be assessed using choroid-level projection OCT images.
Projection OCT fundus imaging facilities rapid interpretation of large 3D-OCT data sets. Projection OCT enhances contrast and visualises outer retinal pathology not visible with standard fundus imaging or OCT fundus imaging. Projection OCT fundus images enable registration with standard ophthalmic diagnostics and cross-sectional OCT images. Outer retinal alterations can be assessed and drusen morphology, photoreceptor impairment and pigmentary abnormalities identified.
PMCID: PMC2743133  PMID: 18662918
20.  Natural History of Cone Disease in the Murine Model of Leber Congenital Amaurosis Due to CEP290 Mutation: Determining the Timing and Expectation of Therapy 
PLoS ONE  2014;9(3):e92928.
Mutations in the CEP290 (cilia-centrosomal protein 290 kDa) gene in Leber congenital amaurosis (LCA) cause early onset visual loss but retained cone photoreceptors in the fovea, which is the potential therapeutic target. A cone-only mouse model carrying a Cep290 gene mutation, rd16;Nrl−/−, was engineered to mimic the human disease. In the current study, we determined the natural history of retinal structure and function in this murine model to permit design of pre-clinical proof-of-concept studies and allow progress to be made toward human therapy. Analyses of retinal structure and visual function in CEP290-LCA patients were also performed for comparison with the results in the model.
Rd16;Nrl−/− mice were studied in the first 90 days of life with optical coherence tomography (OCT), electroretinography (ERG), retinal histopathology and immunocytochemistry. Structure and function data from a cohort of patients with CEP290-LCA (n = 15; ages 7–48) were compared with those of the model.
CEP290-LCA patients retain a central island of photoreceptors with normal thickness at the fovea (despite severe visual loss); the extent of this island declined slowly with age. The rd16;Nrl−/− model also showed a relatively slow photoreceptor layer decline in thickness with ∼80% remaining at 3 months. The number of pseudorosettes also became reduced. By comparison to single mutant Nrl−/− mice, UV- and M-cone ERGs of rd16;Nrl−/− were at least 1 log unit reduced at 1 month of age and declined further over the 3 months of monitoring. Expression of GNAT2 and S-opsin also decreased with age.
The natural history of early loss of photoreceptor function with retained cone cell nuclei is common to both CEP290-LCA patients and the rd16;Nrl−/− murine model. Pre-clinical proof-of-concept studies for uniocular therapies would seem most appropriate to begin with intervention at P35–40 and re-study after one month by assaying interocular difference in the UV-cone ERG.
PMCID: PMC3966841  PMID: 24671090
21.  New developments in murine imaging for assessing photoreceptor degeneration in vivo 
Optical Coherence Tomography (OCT) is a powerful clinical tool that measures near infrared light backscattered from the eye and other tissues. OCT is used for assessing changes in retinal structure, including layer thicknesses, detachments and the presence of drusen in patient populations. Our custom-built OCT system for the mouse eye quantitatively images all layers of the neural retinal, the RPE, Bruchs’ membrane and the choroid. Longitudinal assessment of the same retinal region reveals that the relative intensities of retinal layers are highly stable in healthy tissue, but show progressive increases in intensity in a model of retinal degeneration. The observed changes in OCT signal have been correlated with ultrastructural disruptions that were most dramatic in the inner segments and nuclei of the rods. These early changes in photoreceptor structure coincided with activation of retinal microglia, which migrated vertically from the inner to the outer retina to phagocytose photoreceptor cell bodies (Levine et al. 2014). We conclude that quantitative analysis of OCT light scattering signals may be a useful tool for early detection and subcellular localization of cell stress prior to cell death, and for assessing the progression of degenerative disease over time. Future efforts to develop sensitive approaches for monitoring microglial dynamics in vivo may likewise elucidate earlier signs of cellular stress during retinal degeneration.
PMCID: PMC4603285  PMID: 26427421
22.  Different effects of valproic acid on photoreceptor loss in Rd1 and Rd10 retinal degeneration mice 
Molecular Vision  2014;20:1527-1544.
The histone-deacetylase inhibitor activity of valproic acid (VPA) was discovered after VPA’s adoption as an anticonvulsant. This generated speculation for VPA’s potential to increase the expression of neuroprotective genes. Clinical trials for retinitis pigmentosa (RP) are currently active, testing VPA’s potential to reduce photoreceptor loss; however, we lack information regarding the effects of VPA on available mammalian models of retinal degeneration, nor do we know if retinal gene expression is perturbed by VPA in a predictable way. Thus, we examined the effects of systemic VPA on neurotrophic factor and Nrl-related gene expression in the mouse retina and compared VPA’s effects on the rate of photoreceptor loss in two strains of mice, Pde6brd1/rd1 and Pde6brd10/rd10.
The expression of Bdnf, Gdnf, Cntf, and Fgf2 was measured by quantitative PCR after single and multiple doses of VPA (intraperitoneal) in wild-type and Pde6brd1/rd1 mice. Pde6brd1/rd1 mice were treated with daily doses of VPA during the period of rapid photoreceptor loss. Pde6brd10/rd10 mice were also treated with systemic VPA to compare in a partial loss-of-function model. Retinal morphology was assessed by virtual microscopy or spectral-domain optical coherence tomography (SD-OCT). Full-field and focal electroretinography (ERG) analysis were employed with Pde6brd10/rd10 mice to measure retinal function.
In wild-type postnatal mice, a single VPA dose increased the expression of Bdnf and Gdnf in the neural retina after 18 h, while the expression of Cntf was reduced by 70%. Daily dosing of wild-type mice from postnatal day P17 to P28 resulted in smaller increases in Bdnf and Gdnf expression, normal Cntf expression, and reduced Fgf2 expression (25%). Nrl gene expression was decreased by 50%, while Crx gene expression was not affected. Rod-specific expression of Mef2c and Nr2e3 was decreased substantially by VPA treatment, while Rhodopsin and Pde6b gene expression was normal at P28. Daily injections with VPA (P9–P21) dramatically slowed the loss of rod photoreceptors in Pde6brd1/rd1 mice. At age P21, VPA-treated mice had several extra rows of rod photoreceptor nuclei compared to PBS-injected littermates. Dosing started later (P14) or dosing every second day also rescued photoreceptors. In contrast, systemic VPA treatment of Pde6brd10/rd10 mice (P17–P28) reduced visual function that correlated with a slight increase in photoreceptor loss. Treating Pde6brd10/rd10 mice earlier (P9–P21) also failed to rescue photoreceptors. Treating wild-type mice earlier (P9–P21) reduced the number of photoreceptors in VPA-treated mice by 20% compared to PBS-treated animals.
A single systemic dose of VPA can change retinal neurotrophic factor and rod-specific gene expression in the immature retina. Daily VPA treatment from P17 to P28 can also alter gene expression in the mature neural retina. While daily treatment with VPA could significantly reduce photoreceptor loss in the rd1 model, VPA treatment slightly accelerated photoreceptor loss in the rd10 model. The apparent rescue of photoreceptors in the rd1 model was not the result of producing more photoreceptors before degeneration. In fact, daily systemic VPA was toxic to wild-type photoreceptors when started at P9. However, the effective treatment period for Pde6brd1/rd1 mice (P9–P21) has significant overlap with the photoreceptor maturation period, which complicates the use of the rd1 model for testing of VPA’s efficacy. In contrast, VPA treatment started after P17 did not cause photoreceptor loss in wild-type mice. Thus, the acceleration of photoreceptor loss in the rd10 model may be more relevant where both photoreceptor loss and VPA treatment (P17–P28) started when the central retina was mature.
PMCID: PMC4225157  PMID: 25489226
23.  An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina 
Cell Death & Disease  2014;5(12):e1578-.
Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1β, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1β was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1β. We observed ~19% reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1β.
PMCID: PMC4454166  PMID: 25522272
24.  Systemic Administration of the Antioxidant/Iron Chelator α-Lipoic Acid Protects Against Light-Induced Photoreceptor Degeneration in the Mouse Retina 
Oxidative stress and inflammation have key roles in the light damage (LD) model of retinal degeneration as well as in age-related macular degeneration (AMD). We sought to determine if lipoic acid (LA), an antioxidant and iron chelator, protects the retina against LD.
Balb/c mice were treated with LA or control saline via intraperitoneal injection, and then were placed in constant cool white light-emitting diode (LED) light (10,000 lux) for 4 hours. Retinas were evaluated at several time points after LD. Photoreceptor apoptosis was assessed using the TUNEL assay. Retinal function was analyzed via electroretinography (ERG). Retinal degeneration was assessed after LD by optical coherence tomography (OCT), TUNEL analysis, and histology. The mRNAs of several oxidative stress, inflammation, and iron-related genes were quantified by quantitative PCR (qPCR).
The LD resulted in substantial photoreceptor-specific cell death. Dosing with LA protected photoreceptors, decreasing the numbers of TUNEL-positive photoreceptors and increasing the number of surviving photoreceptors. The retinal mRNA levels of genes indicating oxidative stress, inflammation, and iron accumulation were lower following LD in mice treated with LA than in control mice. The ERG analysis demonstrated functional protection by LA.
Systemic LA is protective against light-induced retinal degeneration. Since this agent already has proven protective in other retinal degeneration models, and is safe and protective against diabetic neuropathy in patients, it is worthy of consideration for a human clinical trial against retinal degeneration or AMD.
The paper shows for the first time to our knowledge that the antioxidant/iron chelator lipoic acid, a widely-available nutritional supplement, protects against light induced retinal degeneration.
PMCID: PMC4172298  PMID: 25146987
lipoic acid; oxidative stress; light damage; retinal degeneration
25.  Optical Coherence Tomography Study of Experimental Anterior Ischemic Optic Neuropathy and Histologic Confirmation 
The optic nerve is part of the central nervous system, and interruption of this pathway due to ischemia typically results in optic atrophy and loss of retinal ganglion cells. In this study, we assessed in vivo retinal changes following murine anterior ischemic optic neuropathy (AION) by using spectral-domain optical coherence tomography (SD-OCT) and compared these anatomic measurements to that of histology.
We induced ischemia at the optic disc via laser-activated photochemical thrombosis, performed serial SD-OCT and manual segmentation of the retinal layers to measure the ganglion cell complex (GCC) and total retinal thickness, and correlated these measurements with that of histology.
There was impaired perfusion and leakage at the optic disc on fluorescein angiography immediately after AION and severe swelling and distortion of the peripapillary retina on day-1. We used SD-OCT to quantify the changes in retinal thickness following experimental AION, which revealed significant thickening of the GCC on day-1 after ischemia followed by gradual thinning that plateaued by week-3. Thickness of the peripapillary sensory retina was also increased on day-1 and thinned chronically. This pattern of acute retinal swelling and chronic thinning on SD-OCT correlated well with changes seen in histology and corresponded to loss of retinal ganglion layer cells after ischemia.
This was a serial SD-OCT quantification of acute and chronic changes following experimental AION, which revealed changes in the GCC similar to that of human AION, but over a time frame of weeks rather than months.
SD-OCT imaging in experimental murine AION revealed the spectrum of anatomic changes following ischemia with initial swelling and gradual thinning out to 3-4 weeks. SD-OCT findings were corroborated by histologic analysis and correlated with gradual loss of retinal ganglion cells over several weeks.
PMCID: PMC3771554  PMID: 23887804
AION; animal model; SD-OCT; retinal ganglion cell; optic neuropathy

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