Visual Function in CRB1-RD
CRB1-RD patients had visual acuities that ranged from 20/30 to LP; more than half of the patients in this cohort (13/21; 62%) had visual acuity of 20/100 or worse (). There was severe retina-wide dysfunction by ERG and perimetry. ERGs were detectable in 14 (67%) of 21 of the patients examined. There were no measurable rod b-waves but there were small cone-mediated signals (). Among the patients with measurable kinetic visual fields, a common pattern was a small, central island of vision separated from a peripheral temporal island by a complete annular midperipheral scotoma; some patients had only peripheral temporal islands as their remnant of vision.
Visual field abnormalities are illustrated in three representative patients (). P4 at age 14 showed a central island and a temporal peripheral island in response to the largest target (V-4e); perception of the small target (I-4e) was limited to the central few degrees. P8 at age 19 had a smaller central island, but there remained a large temporal island of function. P12 at age 28 had a very limited central island near fixation and a temporal field island (A). Rod and cone sensitivity losses across the visual field were mapped for these three patients (B). Consistent with the nondetectable rod ERGs in these and other CRB1-RD patients, the three patients lacked measurable rod function psychophysically across most of the visual field, except for small temporal peripheral islands in each case. Cone sensitivity loss maps show residual but impaired central and temporal peripheral cone islands. P12 has only a central island of cone function detectable. To define in more detail central rod- and cone-mediated function at relatively early disease stages, we measured dark-adapted sensitivity profiles with chromatic stimuli (500 and 650 nm) along the horizontal meridian (central 60°) in the same three patients (C) as well as in seven others (ages, 12–29 years). Even at the youngest ages examined with these stimuli (12 and 13 years), central visual function was abnormal. There was mainly cone-mediated detection of centrally presented stimuli; rod-mediated vision could be measured in loci around fixation in only three patients in the second decade of life and was reduced by at least ~2 log units. Normal cone sensitivity was measurable at the most central locations in a minority of patients; most patients had central islands of cone function, with sensitivity losses ranging from 0.5 to 2.5 log units.
Figure 1. Visual function in representative patients with CRB1-RD. (A) Kinetic perimetry results using two targets (V-4e, I-4e) illustrate preserved central and temporal peripheral islands of vision. (B) Dark-adapted (top) and light-adapted (bottom) static threshold (more ...)
Longitudinal data in P7 for an 11-year period () provided a view of the progression of the disease to the stage with only central and peripheral islands. At age 13, kinetic visual fields with the V-4e target were relatively full in extent except for a scotoma in the inferior nasal field; the I-4e target was perceived only in the central field (A). From ages 15 to 24 years, there was progression of field loss from an incomplete absolute midperipheral scotoma (age 15) to a complete annular scotoma separating a central island from a temporal peripheral island (age 24). Maps of rod and cone sensitivity losses across the visual field (B) or as horizontal sensitivity profiles (C) also demonstrated progression. At age 13, there was a large midperipheral rod and cone scotoma that reached the nasal periphery and surrounded a small central island of reduced rod function but better cone function (B). By age 24, there was only a small island of abnormally reduced cone function with a retained temporal island of reduced (by at least 1.5 log unit) but detectable rod and cone function. Profiles of central visual function across the horizontal meridian (C) indicate that there was severely reduced but detectable extracentral rod function at age 13 (C, top); cone function had near normal sensitivity at fixation (C, bottom). Over the ensuing 11 years, rod function became undetectable and cone function progressively decreased in extent and sensitivity. P15 had longitudinal results spanning a 22-year interval (27–49 years). Perimetric results at age 27 were similar to those of P7 at age 15 but when seen again at age 49, the patient had only hand motions vision, and there was no detection of stimuli in kinetic or static perimetry.
Figure 2. Longitudinal sequence of visual function in a CRB1-RD patient spanning 11 years. (A) Kinetic perimetry results in P7 at three ages. (B) Dark-adapted (top) and light-adapted (bottom) static threshold perimetry results displayed as grayscale maps of rod (more ...)
Retinal and Visual Brain Structure in CRB1-RD
Maps of retinal thickness topography derived from OCT in patients with CRB1
-RD illustrate the structural abnormalities in these patients (). The thickness map of the normal retina, from the retinal pigment epithelium (RPE) to the inner limiting membrane, shows some distinctive features (A, left): a central depression or foveal pit, a surrounding ring of increased thickness with displaced inner retinal layers from foveal formation, a decline in thickness with eccentricity beyond this ring, and a prominent crescent-shaped thickening in the nasal retina extending into the superior and inferior poles of the optic nerve, attributable to the converging axons from ganglion cells. Thickness topography in two representative CRB1
-RD patients (P4 and P12) showed abnormal thickening (at ages 16 and 28); retinal thickness in the central retina was >2 SD from normal mean thickness, except at and near the fovea (A, lower right insets; pink is >2 SD, and white is within normal limits). Vertical cross sections through the fovea (±9 mm; total vertical expanse of ~60°) were analyzed in 19 of the 22 patients (data not shown). There was increased retinal thickness in all but two patients (P7 at ages 26 and 30; P18 at age 45), thus extending our previous observations in this population.5
We also inquired whether there was a change in retinal thickness with age. Serial OCTs (across the vertical meridian, ±9 mm from the fovea) were available in five patients (P2, P4, P7, P8, and P12), each spanning a time interval of at least 4 years. P2 and P4 showed retinal thickness reductions between the ages of 12 and 19 years and 14 and 19 years, respectively. In contrast, P8 and P12 were monitored from ages 18 to 22 and 26 to 30 years, respectively, and there was no decrease in retinal thickness observed. All these patients, whether they showed progressive retinal thinning or not, continued to have hyperthickness at later time points. Of interest, P7, one of two patients with normal retinal thickness, was monitored between ages 24 and 30 years, and retinal thickness remained within normal limits at the later time point.
Figure 3. Retinal and visual pathway structure in CRB1-RD. (A) Topographical maps of retinal thickness in a normal 21-year-old subject (left) and two patients with CRB1-RD. Traces of major blood vessels and location of the ONH are overlaid on each map. Pseudocolor (more ...)
Regions of thickening in CRB1
-RD retina, unlike in the well-laminated normal retina (B, left panel), showed coarse and abnormal layering5
and many intraretinal hyperreflective structures (B, right panels). Hyperreflective lesions could be different in size and could be found at different depths from the vitreoretinal surface (B, arrows and arrowheads). Larger hyperreflective structures could be classified into at least two types based on OCT appearance: One type was associated with a shadowing of signal from deeper layers (B, P8 and P17, long arrows) whereas another type was associated with no detectable shadowing (P6, arrowhead). The shadowing features of smaller hyperreflective lesions were not as certain. Also notable in these scans was the lack of normal photoreceptor layers; and the regions in the retina depicted were associated with little or no measurable vision. We tested the hypothesis that the two types of larger hyperreflective lesions were associated with different en face appearance. OCT scans from nine patients (P2, P4, P5, P6, P10, P12, P15, P16, and P17) were studied to determine the relationship between the en face (SLO) and cross-sectional (OCT) image features. Hyperreflectivity with shadowing was associated with relatively darker lesions on en face images (C, left). These lesions are thought to correspond to pigment migration, including bone-spicule–like pigment and clumped pigmentary changes. Scattering and absorbing properties of melanin would explain the hyperreflectivity and the shadowing. On the other hand, hyperreflectivity without shadowing was associated with no apparent lesions or relatively brighter lesions on en face images (C, right). The basis of these scattering but nonabsorbing OCT features is less clear but could correspond to focal regions of dysplasia and pseudorosettes. Also notable were the hyperreflective abnormalities deep in the retina appearing proximate to the RPE or Bruch's membrane level of the scan (B, P17, short arrows).
The unusually thickened parapapillary nerve fiber layer, we previously observed in CRB1
and the limited literature on visual pathway integrity in LCA with known genotype29
prompted study with MR to determine whether the visual brain is normal in CRB1
-RD (D–F). Visual pathway structures in CRB1
-RD patients appeared normal in MR images. The interpial optic nerve diameter in P8, P12, P11, and P22 (aged 21, 29, 34, and 53, respectively, at the time of the scan) was normal (D), as defined by measurements from our normal subjects and published data.30
A voxel-based morphometric analysis19
of the anatomic images obtained from four CRB1
-RD patients tested whether atrophy was present within the occipital lobe white matter, as has been noted in patients with early-onset blindness of various causes.31
-RD patients fell within the range of control subject data; the CRB1
-RD patient mean, however, was slightly but significantly reduced in comparison to that of the controls (log Jacobian [normed]: controls, 0 ± 0.03 SEM; CRB1
-RD patients, −0.14 ± 0.06; t
] = 2.2, P
= 0.04 one-tailed), indicating relative atrophy of occipital white matter structures. The thickness of the cortical gray matter layer was measured within a striatal region of interest defined by cortical surface topology.21
Increased striatal cortical thickness has been observed in previous studies of early-onset blindness32
and attributed to the failure of developmental synaptic pruning. The gray matter layer was thicker in CRB1
-RD patients than in the controls, but not significantly so (thickness in millimeters: controls, 1.67 ± 0.03 SEM; CRB1
-RD patients, 1.87 ± 0.12; t
] = 1.7; P
= 0.07 one-tailed).
We assessed functional cortical responses to large-field light stimulation.29
Whole-brain responses in CRB1
-RD patients were of reduced amplitude and spatial extent compared with those in normally sighted controls (E). Within the posterior visual cortices (F), the volume of activated tissue was significantly reduced in CRB1
-RD patients compared with that in controls (t
] = 5.4; P
= 0.002 one-tailed). The four CRB1
-RD patients, ranked in order of higher to lower activation volume, were P8, P12, P11, and P22. The severity of retinal disease expression in these patients at the time of fMRI was compared by ranking central visual function. A ranking by visual acuity in the better eye and by dark- and light-adapted visual sensitivity in the central field mirrored the fMRI results, with the least affected being P8 and in order of increasing visual loss: P12, P11, and P22.
Crb1-Mutant rd8 Mice Show Patchy Dysplasia and Some Retinal Degeneration
ERG parameters were compared between rd8 and age-matched WT control mice (A). All rod photoreceptor–driven ERG parameters, except for the dark-adapted maximum b-wave amplitude (Vmax), showed no significant differences between rd8 and WT for all grouped comparisons. For the oldest group of mice, Vmax was significantly smaller than WT: mean (SD) in rd8 =180 (75) μV versus 278 (64) μV in WT (P = 0.002). In addition, one to three rd8 eyes in each group showed responses that fell outside the range recorded in WT eyes. Cone responses elicited with white stimuli were not significantly different from WT (A). Further, responses to UV and green flashes in the light-adapted state did not show significant mismatches in the rd8 group when compared to the WT group (UV-to-green difference: −11.7  μV in rd8, −14.8  μV in WT; P = 0.63), implying normal or near-normal short-wavelength (S) –and middle-wavelength (M)–sensitive cone function in rd8 mice over the age range studied.
Figure 4. Retinal function and histopathology in the Crb1-mutant rd8 mouse. (A) ERG parameters compared in a cohort of WT and rd8 mice of different ages (key for color coding of ages, right). (B) Dorsal-ventral retinal sections in a 4-month-old WT mouse (left) (more ...)
Dorsal-ventral histologic sections through the optic nerve are compared in a WT retina (age 4 months, left) and an rd8
retina (age, 6.5 months, right; B). At low magnification, patches of dysplasia were present in the inferior (ventral) retina of the rd8
mouse (arrows). Retinal sections at higher magnification, from inferior to the optic nerve in the WT and rd8
retinas, are shown adjacent to the full dorsal–ventral sections. The WT section shows the normal architecture with well-defined nuclear layers (GCL, INL, and ONL), synaptic layers, the photoreceptor inner and outer segments, and the RPE. In the rd8
section, the laminae were identifiable, but between the ONL and INL were retinal folds or pseudorosettes, as documented previously in Crb1
Across the rd8
section, there was an apparent variation in ONL and IS/OS thickness.
Sections from the inferior retina of rd8
mice at different ages suggest a possible sequence of abnormalities leading to the dysplasia. Impaired adhesion between photoreceptors and MG cells (due to zonulae adherens junction abnormalities) and loss of OLM integrity have been shown to be early findings in rd8
and other Crb1
-mutant mouse models (for example, Refs. 9
). Displacement of IS/OS material and photoreceptor cell bodies into the inner retina (C, age 2 months) could be the result of fragmentation of the OLM and loss of this normal structural barrier.37
Repair processes that lead to readherence of MG and photoreceptor cells have been postulated to occur,8
and this could lead to isolation of the pseudorosette between INL and ONL (C, 4.5 months). There appear to be variable numbers and sizes of pseudorosettes (B, right; C, 4.5 vs. 6.5 months). This variation may relate to the regional retinal extent of OLM integrity at different disease stages and the modulation of the fragmentation and repair processes by unknown factors.34
This sequence of abnormalities displayed in rd8
mice of increasing age (C, top) was also found in individual animals (C, lower row). A 10-month old rd8
mouse, for example, showed, in inferior (ventral) sections, a similar spectrum of results: ranging from (left to right) no obvious dysplasia, to focal displacement of IS/OS material into ONL, to a formed pseudorosette, to more extensive or multiple pseudorosettes, to an amalgam of ONL and INL and retinal degeneration with reduced ONL thickness and IS/OS.
OCT scans were performed and quantified in 22 rd8
mice and compared with those in 13 WT mice (). OCT cross sections were compared to histologic sections (A) from the inferior retina of WT and rd8
mice. In the WT histology–OCT pair (A, left), the vitreoretinal interface was hyperreflective, and there were hyporeflective zones that represent the INL and ONL; the deep complex hyperreflectivity represents the IS, OS, RPE, and choroid.15
Correspondence between retinal histology and OCT features has been quantified previously.15,38–40
OCT sections also have identifiable vitreoretinal interface hyperreflectivity, INL and ONL hyporeflective layers, and a deep hyperreflective outer retinal complex. At a presumed early stage of pseudorosette formation with IS and OS material displaced into the ONL (see C), there was a hyperreflective region (arrow) with corresponding thinning of the ONL at that locus (A, middle pair). A more fully formed pseudorosette between ONL and INL (A, right pair) is illustrated histologically, and the OCT shows a hyperreflective region (arrowhead) between the INL and ONL with surrounding hyporeflectivity suggestive of the dysplastic lesion.
Figure 5. OCT analysis of the retinal abnormalities in rd8 mice. (A) Histologic and OCT sections in WT (left) and rd8 (middle and right) retinas to illustrate the relationship between the different modalities of determining laminar architecture and how abnormalities (more ...)
Representative vertical OCT scans across the ONH showed that there were hyperreflective abnormalities between the INL and ONL at all ages studied in the rd8
animals (B). All rd8
animals between the ages of 3 and 7 months (n
= 11) showed abnormal hyperreflective structures in the OPL region (arrowheads), and some also had hyperreflective lesions that spanned the retina from the deep outer retinal complex into the ONL (arrow). Of the 10-month old rd8
eyes, eight of nine showed such hyperreflectivity in the inferior retina and four of nine eyes showed similar abnormalities superiorly. The dramatic retinal thickening in human CRB1
prompted us to analyze retinal thickness in the rd8
retinas. Retinal thickness measurements of OCT vertical scans from each of three rd8
age groups did not show remarkable differences compared with WT data (C). There was a reduction of rd8
inferior retina thickness with age, whereas the superior retinas were relatively constant.
Prompted by the relatively normal ERGs in the rd8 mice, we asked whether there was any evidence of outer retinal abnormalities, specifically the IS+OS thickness (D). ONL integrity, compromised by pseudorosette formation, was a less feasible target for morphometry. We compared IS+OS thickness in rd8 (n = 4, one eye per animal, ages 4 and 6.5 months) and WT (n = 2, one eye per animal, age 4 months) mice. Eight histologic sections were used for each eye. Within each section, a single pair of measurements was performed at adjacent locations. In rd8 eyes, adjacent areas with and without dysplastic changes (D, inset) were compared. There was a significantly thinner IS+OS in dysplastic regions (P = 0.023, ANOVA), and the mean difference from neighboring nondysplastic regions was 5.1 μm or 20% (D). There was no significant effect of age (P = 0.99, ANOVA), and there were no significant differences between WT eyes and nondysplastic regions of rd8 eyes (P = 0.73, ANOVA).