Inner Segment Research Articles

Restricted Spots of Light Reveal an Efficacy Gradient of the Phototransduction Cascade Along the Rod Outer Segment

Rod photoreceptors consist of an outer segment (OS) and an inner segment (IS). Inside the OS a biochemical machinery transforms the absorption of photons into electrical signals (Yoshizawa and Wald, 1963; Baylor et al., 1979; Fung and Strayer, 1980). This biochemical machinery has been treated and thought to be in essence homogenous within OS with only marginal dishomogeneities (Lamb et al., 1981; Schnapf, 1983; Jarvinen and Lamb, 2005). In order to verify this assumption, we have used special optical fibres to deliver highly-localized light stimuli to OS. If a Gaussian beam of light is fed into an apertureless tapered optical fibers (TOF), coated with gold and titanium, the exiting spot of light does not have the usual lobes and is fairly restricted. As the TOF is moved from the OS base towards its tip, the amplitude of both the saturating response and the single-photon response decreased, demonstrating a pronounced reduction in the efficacy of the phototransduction machinery. We propose that this reduction in efficacy reflects a reduction in PDE levels along the OS, associated with discs aging. From measurements of the gradient of the saturating responses elicited by local flashes at different positions, we calculate that the longitudinal diffusion coefficient for cyclic guanosine monophosphate (cGMP) exceeds 500 µm2/s. As a result of this high diffusion coefficient, cyclic nucleotide-gated (CNG) channels near the tip of the outer segment are closed by diffuse flashes of light, despite the very low level of PDE in the discs in that region.

Macular Cone Abnormalities in Retinitis Pigmentosa with Preserved Central Vision Using Adaptive Optics Scanning Laser Ophthalmoscopy

PurposeTo assess macular photoreceptor abnormalities in eyes with retinitis pigmentosa (RP) with preserved central vision using adaptive optics scanning laser ophthalmoscopy (AO-SLO).MethodsFourteen eyes of 14 patients with RP (best-corrected visual acuity 20/20 or better) and 12 eyes of 12 volunteers underwent a full ophthalmologic examination, fundus autofluorescence, spectral-domain optical coherence tomography (SD-OCT), and imaging with a prototype AO-SLO system. Cone density and spatial organization of the cone mosaic were assessed using AO-SLO images.ResultsIn 3 eyes with RP and preserved central vision, cones formed a mostly regular mosaic pattern with small patchy dark areas, and in 10 eyes, the cone mosaic patterns were less regular, and large dark regions with missing cones were apparent. Only one eye with RP demonstrated a normal, regular cone mosaic pattern. In eyes with RP, cone density was significantly lower at 0.5 mm and 1.0 mm from the center of the fovea compared to normal eyes (P<0.001 and 0.021, respectively). At 0.5 mm and 1.0 mm from the center of the fovea, a decreased number of cones had 6 neighbors in eyes with RP (P = 0.002 for both). Greater decrease in cone density was related to disruption of the photoreceptor inner segment (IS) ellipsoid band on SD-OCT images (P = 0.044); however, dark regions were seen on AO-SLO even in areas of continuous IS ellipsoid on SD-OCT. Decreased cone density correlated thinner outer nuclear layer (P = 0.029) and thinner inner segment and outer segment thickness (P = 0.011) on SD-OCT.ConclusionsCone density is decreased and the regularity of the cone mosaic spatial arrangement is disrupted in eyes with RP, even when visual acuity and foveal sensitivity are good. AO-SLO imaging is a sensitive quantitative tool for detecting photoreceptor abnormalities in eyes with RP.

IQCB1andPDE6BMutations Cause Similar Early Onset Retinal Degenerations in Two Closely Related Terrier Dog Breeds

To identify the causative mutations in two early-onset canine retinal degenerations, crd1 and crd2, segregating in the American Staffordshire terrier and the Pit Bull Terrier breeds, respectively. Retinal morphology of crd1- and crd2-affected dogs was evaluated by light microscopy. DNA was extracted from affected and related unaffected controls. Association analysis was undertaken using the Illumina Canine SNP array and PLINK (crd1 study), or the Affymetrix Version 2 Canine array, the "MAGIC" genotype algorithm, and Fisher's Exact test for association (crd2 study). Positional candidate genes were evaluated for each disease. Structural photoreceptor abnormalities were observed in crd1-affected dogs as young as 11-weeks old. Rod and cone inner segment (IS) and outer segments (OS) were abnormal in size, shape, and number. In crd2-affected dogs, rod and cone IS and OS were abnormal as early as 3 weeks of age, progressing with age to severe loss of the OS, and thinning of the outer nuclear layer (ONL) by 12 weeks of age. Genome-wide association study (GWAS) identified association at the telomeric end of CFA3 in crd1-affected dogs and on CFA33 in crd2-affected dogs. Candidate gene evaluation identified a three bases deletion in exon 21 of PDE6B in crd1-affected dogs, and a cytosine insertion in exon 10 of IQCB1 in crd2-affected dogs. Identification of the mutations responsible for these two early-onset retinal degenerations provides new large animal models for comparative disease studies and evaluation of potential therapeutic approaches for the homologous human diseases.

Morphologic Characteristics of the Outer Retina in Cone Dystrophy on Spectral-domain Optical Coherence Tomography

PurposeTo investigate the morphologic changes in the outer retina of patients with cone dystrophy, using spectral-domain optical coherence tomography (SD-OCT).MethodsThe medical records of 15 cone dystrophy patients examined from January 2007 to January 2012 were reviewed retrospectively. All patients underwent ophthalmic evaluation including best-corrected visual acuity (BCVA), color vision testing, fundus examination, full-field standard electroretinography (ERG), multifocal (mf) ERG, and SD-OCT. Qualitative and quantitative SD-OCT data and ERG responses were analyzed and compared among the patient categories and the normal control group.ResultsThere were 4 major categories of SD-OCT findings, based on the status of the ellipsoid portion of the photoreceptor inner segment (ISe), outer segment (OS) contact cylinder, and retinal pigment epithelium (RPE) layer. Category 0 showed no structural abnormalities. Category 1 showed foveal ISe loss and obscurity of the border between the ISe band and the external limiting membrane (ELM). Category 2 showed foveal thinning and focal foveal ISe disruption with an intact ELM. Category 3 showed foveal thickening and perifoveal disruption of the ISe layer. Category 1 to 3 showed OS contact cylinder layer absence and RPE thickening. The patients in category 0 tended to be younger (mean, 10.0 years) than those in categories 1 to 3 (mean, 17.6 years), although this difference was not statistically significant. Category 1 to 3 patients exhibited statistically significant thinning of the central retina and outer nuclear layer and thickening of the RPE layer relative to the category 0 and normal control group. There was a significant correlation between the central foveal thickness and BCVA in the patients with cone dystrophy. ERG and mfERG responses did not differ significantly among the different cone dystrophy categories.ConclusionsThe morphologic features of cone dystrophy as revealed by SD-OCT, could be categorized as either normal or 1 of 3 different types of outer retinal changes. The presence of normal retinal structures in young cone dystrophy patients with functional impairment (category 0) indicates that electrophysiologic studies are superior to current imaging modalities for the early diagnosis of cone dystrophy. The characteristic SD-OCT findings in cone dystrophy patients may aid in differential diagnosis and be useful for future research on the pathology of cone dystrophy.

Immunocytochemical Evidence of the Localization of the Crumbs Homologue 3 Protein (CRB3) in the Developing and Mature Mouse Retina

CRB3 (Crumbs homologue 3), a member of the CRB protein family (homologous to the Drosophila Crumbs), is expressed in different epithelium-derived cell types in mammals, where it seems to be involved in regulating the establishment and stability of tight junctions and in ciliogenesis. This protein has been also detected in the retina, but little is known about its localization and function in this tissue. Our goal here was to perform an in-depth study of the presence of CRB3 protein in the mouse retina and to analyze its expression during photoreceptor ciliogenesis and the establishment of the plexiform retinal layers. Double immunofluorescence experiments for CRB3 and well-known markers for the different retinal cell types were performed to study the localization of the CRB3 protein. According to our results, CRB3 is present from postnatal day 0 (P0) until adulthood in the mouse retina. It is localized in the inner segments (IS) of photoreceptor cells, especially concentrated in the area where the connecting cilium is located, in their synaptic terminals in the outer plexiform layer (OPL), and in sub-populations of amacrine and bipolar cells in the inner plexiform layer (IPL).

Three-Dimensional Architecture of the Rod Sensory Cilium and Its Disruption in Retinal Neurodegeneration

Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

Ultrastructure of the distal retina of the adult zebrafish, Danio rerio

The organization, morphological characteristics, and synaptic structure of photoreceptors in the adult zebrafish retina were studied using light and electron microscopy. Adult photoreceptors show a typical ordered tier arrangement with rods easily distinguished from cones based on outer segment (OS) morphology. Both rods and cones contain mitochondria within the inner segments (IS), including the large, electron-dense megamitochondria previously described (Kim et al.) Four major ultrastructural differences were observed between zebrafish rods and cones: (1) the membranes of cone lamellar disks showed a wider variety of relationships to the plasma membrane than those of rods, (2) cone pedicles typically had multiple synaptic ribbons, while rod spherules had 1–2 ribbons, (3) synaptic ribbons in rod spherules were ∼2 times longer than ribbons in cone pedicles, and (4) rod spherules had a more electron-dense cytoplasm than cone pedicles. Examination of photoreceptor terminals identified four synaptic relationships at cone pedicles: (1) invaginating contacts postsynaptic to cone ribbons forming dyad, triad, and quadrad synapses, (2) presumed gap junctions connecting adjacent postsynaptic processes invaginating into cone terminals, (3) basal junctions away from synaptic ribbons, and (4) gap junctions between adjacent photoreceptor terminals. More vitread and slightly farther removed from photoreceptor terminals, extracellular microtubule-like structures were identified in association with presumed horizontal cell processes in the OPL. These findings, the first to document the ultrastructure of the distal retina in adult zebrafish, indicate that zebrafish photoreceptors have many characteristics similar to other species, further supporting the use of zebrafish as a model for the vertebrate visual system.

Real-time imaging of rabbit retina with retinal degeneration by using spectral-domain optical coherence tomography.

BackgroundRecently, a transgenic rabbit with rhodopsin Pro 347 Leu mutation was generated as a model of retinitis pigmentosa (RP), which is characterized by a gradual loss of vision due to photoreceptor degeneration. The purpose of the current study is to noninvasively visualize and assess time-dependent changes in the retinal structures of a rabbit model of retinal degeneration by using speckle noise-reduced spectral-domain optical coherence tomography (SD-OCT).Methodology/Principal FindingsWild type (WT) and RP rabbits (aged 4–20 weeks) were investigated using SD-OCT. The total retinal thickness in RP rabbits decreased with age. The thickness of the outer nuclear layer (ONL) and between the external limiting membrane and Bruch's membrane (ELM–BM) were reduced in RP rabbits around the visual streak, compared to WT rabbits even at 4 weeks of age, and the differences increased with age. However, inner nuclear layer (INL) thickness in RP rabbits did not differ from that of WT during the observation period. The ganglion cell complex (GCC) thickness in RP rabbits increased near the optic nerve head but not around the visual streak in the later stages of the observation period. Hyper-reflective change was widely observed in the inner segments (IS) and outer segments (OS) of the photoreceptors in the OCT images of RP rabbits. Ultrastructural findings in RP retinas included the appearance of small rhodopsin-containing vesicles scattered in the extracellular space around the photoreceptors.Conclusions/SignificanceIn the current study, SD-OCT provided the pattern of photoreceptor degeneration in RP rabbits and the longitudinal changes in each retinal layer through the evaluation of identical areas over time. The time-dependent changes in the retinal structure of RP rabbits showed regional and time-stage variations. In vivo imaging of RP rabbit retinas by using SD-OCT is a powerful method for characterizing disease dynamics and for assessing the therapeutic effects of experimental interventions.

Investigation of the hyper-reflective inner/outer segment band in optical coherence tomography of living frog retina

This study is to test anatomic correlates, including connecting cilium (CC) and inner segment (IS) ellipsoid, to the hyper-reflective band visualized by optical coherence tomography (OCT) and commonly attributed to the photoreceptor inner/outer segment (IS/OS) junction. A line-scan OCT (LS-OCT) was constructed to achieve sub-cellular resolution (lateral: ≈ 2 μm; axial: ≈ 4 μm) of excised living frog retinas. An electro-optic phase modulator was employed for rapid and vibration-free phase modulation. Comparison of normalized distance measurements between LS-OCT images and histological images revealed that the dominant source of the signal reported as the IS/OS OCT band actually originates from the IS.

Expression of p97/VCP and ubiquitin during postnatal development of the degenerating rat retina

In this study, we aimed to investigate the distribution pattern of ubiquitin and p97/VCP in the rat retina during postnatal development. Eyeballs from 1-, 4-, 10-, 36- and 72-week-old rats were examined by immunohistochemistry, and protein colocalization was determined by immunofluorescence microscopy. In the 1-week-old rat retina, p97/VCP was strongly expressed in the neuroblast layer, however no ubiquitin immunoreactivity was observed. p97/VCP immunoreactivity was present in the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment (IS) of the photoreceptor layer, and retinal pigment epithelium in the 4- and 10-week-old rat retinas. p97/VCP immunoreactivity increased significantly in the 10-week-old rat retinas. Ubiquitin was barely seen in the 4-week-old rat retinas, and ubiquitin expression was weak in the GCL and the IPL of the 10-week-old rat retinas. In the 36- and 72-week-old rats, the presence of ubiquitin was remarkable in the IS, INL, IPL and GCL, however, p97/VCP immunoreactivity was significantly decreased. Colocalization of ubiquitin and p97/VCP was also observed in the INL, IS, GCL and ONL of 36- and 72-week-old rat retinas. Our results indicate that p97/VCP immunoreactivity in the retina significantly decreases after rats reach 10weeks of age, whereas ubiquitin immunoreactivity increases with aging. These results suggest that an altered expression pattern of p97/VCP and ubiquitin in the developing rat retina may associate with age-related retinal degeneration.

Effects of Age, Sex, and Axial Length on the Three-Dimensional Profile of Normal Macular Layer Structures

To identify sex-related differences and age-related changes in individual retinal layer thicknesses in a population of healthy eyes across the lifespan, using spectral domain optical coherence tomography (SD-OCT). In seven institutes in Japan, mean thicknesses of the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor inner segment (IS), and photoreceptor outer segment (OS) were measured using SD-OCT with a new automated segmentation protocol in 256 healthy subjects. Interoperator coefficients of variability for measurements of each layer ranged from 0.012 to 0.038. The RNFL, GCL, IPL, and INL were thinnest in the foveal area, whereas the OPL+ONL and OS were thickest in this area. Mean thicknesses of the INL and the OPL+ONL were significantly greater in men (P = 0.002 and 0.001, respectively). However, mean RNFL thickness was greater in women (P = 0.006). Thicknesses of the RNFL, GCL, IPL, INL, and IS correlated negatively with age. Thickness of the OPL+ONL was not correlated with age, and thickness of the OS correlated positively with age. Inner retinal (RNFL+GCL+IPL) thickness over the whole macula correlated negatively with age (P < 0.001), but outer retinal (OPL+ONL+IS+OS) thickness did not. Thicknesses of layers did not correlate with axial length. Macular layer thicknesses measured on SD-OCT images in healthy eyes showed significant variations by sex and age. These findings should inform macular layer thickness analyses in SD-OCT studies of retinal diseases and glaucoma.

In SituVisualization of Protein Interactions in Sensory Neurons: Glutamic Acid-Rich Proteins (GARPs) Play Differential Roles for Photoreceptor Outer Segment Scaffolding

Vertebrate photoreceptors initiate vision via a G-protein-mediated signaling cascade organized within a specialized cilium, the outer segment (OS). The membranous "stacked pancake" architecture of this organelle must be partially renewed daily to maintain cell function and viability; however, neither its static structure nor renewal process is well described in molecular terms. Glutamic acid-rich proteins (GARPs), including the cyclic nucleotide-gated cation channel (CNGB1) and GARP2 (a CNGB1 splice-variant), are proposed to contribute to OS organization in concert with peripherin/rds (P/rds), a retinal tetraspanin. We developed and applied an in situ fluorescence complementation approach that offers an unprecedented glimpse at the formation, trafficking, and localization of GARP-P/rds interactions in transgenic Xenopus laevis rod photoreceptors. Interactions for these (and other) proteins could be readily visualized using confocal microscopy. Nearly all associations, including CNGB1-P/rds interaction, were initiated within inner segments (ISs) before trafficking to OSs. In contrast, GARP2-P/rds interactions were only observed downstream, at or near sites of disk morphogenesis. These results suggest that GARP2-P/rds interaction participates directly in structuring disk stacks but CNGB1-P/rds interaction does not and instead serves mainly to localize plasma membrane ion channels. Altogether, the results lead us to propose that differential interaction of GARPs with P/rds may contribute to the broad phenotypic heterogeneity produced by inherited defects in P/rds. Analogous experiments applied to the synaptic protein RIBEYE suggest that monomers can oligomerize at the level of the IS before ribbon assembly and demonstrate the general applicability of this strategy for in situ analysis of protein interactions in sensory neurons.

Automated segmentation of outer retinal layers in macular OCT images of patients with retinitis pigmentosa

To provide a tool for quantifying the effects of retinitis pigmentosa (RP) seen on spectral domain optical coherence tomography images, an automated layer segmentation algorithm was developed. This algorithm, based on dual-gradient information and a shortest path search strategy, delineates the inner limiting membrane and three outer retinal boundaries in optical coherence tomography images from RP patients. In addition, an automated inner segment (IS)/outer segment (OS) contour detection method based on the segmentation results is proposed to quantify the locus of points at which the OS thickness goes to zero in a 3D volume scan. The segmentation algorithm and the IS/OS contour were validated with manual segmentation data. The segmentation and IS/OS contour results on repeated measures showed good within-day repeatability, while the results on data acquired on average 22.5 months afterward demonstrated a possible means to follow disease progression. In particular, the automatically generated IS/OS contour provided a possible objective structural marker for RP progression.

Integrity of the Cone Photoreceptor Mosaic in Oligocone Trichromacy

Oligocone trichromacy (OT) is an unusual cone dysfunction syndrome characterized by reduced visual acuity, mild photophobia, reduced amplitude of the cone electroretinogram with normal rod responses, normal fundus appearance, and normal or near-normal color vision. It has been proposed that these patients have a reduced number of normal functioning cones (oligocone). This paper has sought to evaluate the integrity of the cone photoreceptor mosaic in four patients previously described as having OT. Retinal images were obtained from two brothers (13 and 15 years) and two unrelated subjects, one male (47 years) and one female (24 years). High-resolution images of the cone mosaic were obtained using high-speed adaptive optics (AO) fundus cameras. Visible structures were analyzed for density using custom software. Additional retinal images were obtained using spectral domain optical coherence tomography (SD-OCT), and the four layers of the photoreceptor-retinal pigment epithelium complex (ELM, IS/OS, RPE1, RPE2) were evaluated. Cone photoreceptor length and the thickness of intraretinal layers were measured and compared to previously published normative data. The adult male subject had infantile onset nystagmus while the three other patients did not. In the adult male patient, a normal appearing cone mosaic was observed. However, the three other subjects had a sparse mosaic of cones remaining at the fovea, with no structure visible outside the central fovea. On SD-OCT, the adult male subject had a very shallow foveal pit, with all major retinal layers being visible, and both inner segment (IS) and outer segment (OS) length were within normal limits. In the other three patients, while all four layers were visible in the central fovea and IS length was within normal limits, the OS length was significantly decreased. Peripherally the IS/OS layer decreased in intensity, and the RPE1 layer was no longer discernable, in keeping with the lack of cone structure observed on AO imaging outside the central fovea. Findings are consistent with the visual deficits being caused by a reduced number of healthy cones in the two brothers and the adult female. In the unrelated adult subject, no structural basis for the disorder was found. These data suggest two distinct groups on the basis of structural imaging. It is proposed that the former group with evidence of a reduction in cone numbers is more in keeping with typical OT, with the latter group representing an OT-like phenotype. These two groups may be difficult to readily discern on the basis of phenotypic features alone, and high-resolution imaging may be an effective way to distinguish between these phenotypes.

Extramitochondrial tricarboxylic acid cycle in retinal rod outer segments

Vertebrate retinal rod Outer Segments (OS) are the site of visual transduction, an energy demanding process for which mechanisms of ATP supply are still poorly known. Glycolysis or diffusion of either ATP or phosphocreatine from the Inner Segment (IS) does not seem to display adequate timing to supply ATP for phototransduction. We have previously reported data suggesting an aerobic metabolism in OS, which would largely account for the light-stimulated ATP need of the photoreceptor. Here, by oxymetry and biochemical analyses we show that: (i) disks isolated by Ficoll flotation consume O 2 in the presence of physiological respiring substrates either in coupled or uncoupled conditions; (ii) OS homogenates contain the whole biochemical machinery for the degradation of glucose, i.e. glycolysis and the tricarboxylic acid cycle (TCA cycle), consistently with the results of our previous proteomic study. Activities of the 8 TCA cycle enzymes in OS were comparable to those in retinal mitochondria-enriched fractions. Disk and OS preparations were subjected to TEM analysis, and while they can be considered free of inner segment contaminants, immunogold with specific antibodies demonstrate the expression therein of both the visual pigment rhodopsin and F oF 1-ATP synthase. Finally, double immunofluorescence on mouse retina sections demonstrated a colocalization of some respiratory complex mitochondrial proteins with rhodopsin in rod OS. Data, suggestive of the exportability of the mitochondrial machinery for aerobic metabolism, may shed light on those retinal pathologies related to energy supply impairment in OS and to mutations in TCA enzymes.

Predictors of Short-Term Visual Outcome after Anti-VEGF Therapy of Macular Edema due to Central Retinal Vein Occlusion

The purpose of this study was to analyze predictive factors for best-corrected visual acuity (BCVA) after anti-VEGF treatment in patients with macular edema (ME) secondary to central retinal vein occlusion (CRVO). This prospective study enrolled treatment-naive patients with ME secondary to CRVO. BCVA, ophthalmoscopy, fundus photography, and spectral domain optical coherence tomography (SD-OCT) imaging were performed. SD-OCT was analyzed for integrity of the external limiting membrane (ELM), photoreceptor inner segments (IS), and outer segments (OS). Patients were treated with intravitreal bevacizumab (1.25 mg) or ranibizumab (0.5 mg). BCVA outcome was analyzed 4 weeks after the first injection. Sixty-two eyes of 62 patients (39 men, 23 women; mean age: 67 ±16 years) were included. In 55%, the ELM was intact. These eyes also showed intact photoreceptor IS/OS in horizontal and vertical single scans. Disturbed ELM was seen in 45% and was accompanied by focal disintegration of IS/OS. Four weeks after injection, 58% showed clinically relevant increases of BCVA (≥5 letters). Mean BCVA ranged from 20 to 86 letters. The mean BCVA increase was 18 ± 12 letters in eyes with intact ELM compared with 4 ± 10 letters with disturbed ELM (P < 0.001). Depending on the integrity of the outer retinal layers, the authors observed rapid and clinically relevant improvement in BCVA after the first anti-VEGF injection. In the development of an optimal treatment regime, the indication for treatment and re-treatment should be based on functional and morphologic findings, such as the deterioration of outer retinal layers. Intact ELM in SD-OCT imaging is associated with better visual outcomes after intravitreal anti-VEGF treatment in patients with ME secondary to CRVO.

Method for deriving visual field boundaries from OCT scans of patients with retinitis pigmentosa

The location of the loss of the inner segment (IS)/outer segment (OS) border, as seen with frequency domain optical coherence tomography (fdOCT), was determined on fdOCT scans from patients with retinitis pigmentosa. A comparison to visual field loss supported the hypothesis, based upon previous work, that the point at which the IS/OS border disappears provides a structural marker for the edge of the visual field. Repeat fdOCT measures showed good within day reproducibility, while data obtained on average 22.5 months later showed signs of progression. The IS/OS contour shows promise as a measure for following changes in patients undergoing treatment.

Salamander retina phospholipids and their localization by MALDI imaging mass spectrometry at cellular size resolution

Salamander large cells facilitated identification and localization of lipids by MALDI imaging mass spectrometry. Salamander retina lipid extract showed similarity with rodent retina lipid extract in phospholipid content and composition. Like rodent retina section, distinct layer distributions of phospholipids were observed in the salamander retina section. Phosphatidylcholines (PCs) composing saturated and monounsaturated fatty acids (PC 32:0, PC 32:1, and PC 34:1) were detected mainly in the outer and inner plexiform layers (OPL and IPL), whereas PCs containing polyunsaturated fatty acids (PC 36:4, PC 38:6, and PC 40:6) composed the inner segment (IS) and outer segment (OS). The presence of PCs containing polyunsaturated fatty acids in the OS layer implied that these phospholipids form flexible lipid bilayers, which facilitate phototransduction process occurring in the rhodopsin rich OS layer. Distinct distributions and relative signal intensities of phospholipids also indicated their relative abundance in a particular cell or a cell part. Using salamander large cells, a single cell level localization and identification of biomolecules could be achieved by MALDI imaging mass spectrometry.

Temporal and spatial characteristics of cone degeneration in RCS rats

The temporal and spatial characteristics of cone degeneration in the Royal College of Surgeons (RCS) rat were studied to provide information for treatment strategies of retinitis pigmentosa. Nonpigmented dystrophic RCS rats (RCS) and pigmented nondystrophic RCS rats (controls) were used. Cone processes were visualized with peanut agglutinin (PNA). Cone development appears to have been completed by postnatal day 21 (P21) in both the RCS and control rats. Signs of cone degeneration were obvious by P30, with shorter outer segments (OSs) and enlarged inner segments (ISs). At that time, 81.7% of the cones retained stained ISs. The rate of IS density decline was slower in the peripheral, nasal, and superior retina, and only 43.6% of the cones with ISs were present at P45. By P60, PNA-labeled cone ISs were distorted and restricted to the peripheral retina, and by P90, few cone pedicles were detected. Our findings indicate that therapeutic strategies aimed at rescuing cones in the degenerating retina should be applied before P21 and no later than P45 while substantial numbers of cones retain their ISs. Either the middle or peripheral regions of the nasal and superior retina are the best locations for transplantation strategies.

Suppression and Replacement Gene Therapy for Autosomal Dominant Disease in a Murine Model of Dominant Retinitis Pigmentosa

For dominantly inherited disorders development of gene therapies, targeting the primary genetic lesion has been impeded by mutational heterogeneity. An example is rhodopsin-linked autosomal dominant retinitis pigmentosa with over 150 mutations in the rhodopsin gene. Validation of a mutation-independent suppression and replacement gene therapy for this disorder has been undertaken. The therapy provides a means of correcting the genetic defect in a mutation-independent manner thereby circumventing the mutational diversity. Separate adeno-associated virus (AAV) vectors were used to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site. Viruses were subretinally coinjected into P347S mice, a model of dominant rhodopsin-linked retinitis pigmentosa. Benefit in retinal function and structure detected by electroretinography (ERG) and histology, respectively, was observed for at least 5 months. Notably, the photoreceptor cell layer, absent in 5-month-old untreated retinas, contained 3–4 layers of nuclei, whereas photoreceptor ultrastructure, assessed by transmission electron microscopy (TEM) improved significantly. The study provides compelling evidence that codelivered suppression and replacement is beneficial, representing a significant step toward the clinic. Additionally, dual-vector delivery of combined therapeutics represents an exciting approach, which is potentially applicable to other inherited disorders.