[Show abstract][Hide abstract] ABSTRACT: Purpose:
We investigated the interplay between photoreceptors expressing mutant ELOVL4 (responsible for Stargardt-like disease, STGD3) and RPE in the initial stages of retinal degeneration.
Using electron microscopy and electroretinogram (ERG), we assessed RPE and photoreceptor ultrastructure and function in transgenic ELOVL4 (TG1-2 line; TG) and wild-type (WT) littermates. Experiments were done at P30, 1 month before photoreceptor loss in TG and at P90, a time point with approximately 30% rod loss. To further elucidate the mechanism underlying our ultrastructural and functional results, we undertook Western blotting and immunohistochemistry of key proteins involved in phagocytosis of outer segments by RPE cells.
Firstly, we showed that in TG mouse photoreceptors, endogenous ELOVL4 protein is not mislocalized in the presence of the mutated ELOVL4 protein. Secondly, we found evidence of RPE toxicity at P30, preceding any photoreceptor loss. Pathology in RPE cells was exacerbated at P90. Furthermore, higher proportions of phagosomes remained at the apical side of RPE cells. Subretinal lysosomal deposits were immunopositive for phagocytic proteins. Ultrastructural analysis of photoreceptor (rod) outer segments showed disrupted surface morphology consisting of disc spacing irregularities. Finally, rods and RPE exhibited signs of dysfunction as measured by the ERG a-wave leading edge (P30) and c-wave (P90), respectively.
The presence of human mutant ELOVL4 in transgenic mouse photoreceptors leads to early outer segment disc pathology and RPE cytotoxicity. Defective processing of these abnormal discs by RPE cells ultimately may be responsible for outer segment truncation, photoreceptor death, and vision loss.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
The P23H rhodopsin mutation is an autosomal dominant cause of retinitis pigmentosa (RP). The degeneration can be tracked using different anatomical and functional methods. In our case, we evaluated the anatomical changes using Spectral-Domain Optical Coherence Tomography (SD-OCT) and correlated the findings with retinal thickness values determined by immunocytochemistry.
Pigmented rats heterozygous for the P23H mutation, with ages between P18 and P180 were studied. Function was assessed by means of optomotor testing and ERGs. Retinal thicknesses measurements, autofluorescence and fluorescein angiography were performed using Spectralis OCT. Retinas were studied by means of immunohistochemistry.
Between P30 and P180, visual acuity decreased from 0.500 to 0.182 cycles per degree (cyc/deg) and contrast sensitivity decreased from 54.56 to 2.98 for a spatial frequency of 0.089 cyc/deg. Only cone-driven b-wave responses reached developmental maturity. Flicker fusions were also comparable at P29 (42 Hz). Double flash-isolated rod-driven responses were already affected at P29. Photopic responses revealed deterioration after P29.A reduction in retinal thicknesses and morphological modifications were seen in OCT sections. Statistically significant differences were found in all evaluated thicknesses. Autofluorescence was seen in P23H rats as sparse dots. Immunocytochemistry showed a progressive decrease in the outer nuclear layer (ONL), and morphological changes. Although anatomical thickness measures were significantly lower than OCT values, there was a very strong correlation between the values measured by both techniques.
In pigmented P23H rats, a progressive deterioration occurs in both retinal function and anatomy. Anatomical changes can be effectively evaluated using SD-OCT and immunocytochemistry, with a good correlation between their values, thus making SD-OCT an important tool for research in retinal degeneration.
Frontiers in Neuroanatomy 12/2014; 8:151. DOI:10.3389/fnana.2014.00151 · 3.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose:
We recorded oscillatory potentials (OPs) to document how age impacts on rod- and cone-driven inner retina function.
Dark- and light-adapted electroretinogram (ERG) luminance-response functions were recorded in healthy human subjects aged 20 to 39, 40 to 59, and 60 to 82 years. Raw ERG traces (0.1-300 Hz) were filtered (75-300 Hz) to measure OPs trough-to-peak in the time-amplitude domain. Morlet wavelet transform (MWT) allowed documenting OPs time-amplitude-frequency distribution from raw traces.
Under dark adaptation, both methods revealed reduced OP amplitudes and prolonged implicit times by 40 years of age. The MWT identified a high-frequency band as the main oscillator, which frequency (150-155 Hz) was unaffected by age. Under light adaptation, most OP peaks were delayed by 40 years of age. Peak-trough measures yielded inconsistent results in relation to luminance. Contrastingly, MWT distinguished two frequency bands at all luminances: high frequency (135 ± 6 Hz) time locked to the onset of early OPs and low frequency (82 ± 7 Hz), giving rise to early and late OPs. By 60 years, there was a consistent power reduction specific to the low-frequency band.
Age-related OP changes precede those seen with a- (photoreceptoral) and b-waves (postphotoreceptoral). In addition, MWT allows quantifying distinct low- and high-frequency oscillators in the human retina, which complement traditional OP analysis methods. The identification of an age-independent OP marker (light-adapted high frequency band) opens a new dimension for the screening of retinal degenerations and their impact on inner retina function.
[Show abstract][Hide abstract] ABSTRACT: We sought to study the expression pattern of Disabled-1 (Dab1; an adaptor protein in the reelin pathway) in the cone-rich retina of a diurnal murine rodent. Expression was examined by western blotting and immunohistochemistry using well-established antibodies against Dab1 and various markers of retinal neurons. Western blots revealed the presence of Dab1 (80 kDa) in brain and retina of the Nile grass rat. Retinal immunoreactivity was predominant in soma and dendrites of horizontal cells as well as in amacrine cell bodies aligned at the INL/IPL border. Dab1(+) neurons in the inner retina do not stain for parvalbumin, calbindin, protein kinase C-alpha, choline acetyltransferase, glutamic acid decarboxylase, or tyrosine hydroxylase. They express, however, the glycine transporter GlyT1. They have small ovoid cell bodies (7.1±1.06 μm in diameter) and bistratified terminal plexii in laminas a and b of the IPL. Dab1(+) amacrine cells are evenly distributed across the retina (2600 cells/mm(2)) in a fairly regular mosaic (regularity indexes ≈3.3-5.5). We conclude that retinal Dab1 in the adult Nile grass rat exhibits a dual cell patterning similar to that found in human. It is expressed in horizontal cells as well as in a subpopulation of glycinergic amacrine cells undetectable with antibodies against calcium-binding proteins. These amacrine cells are likely of the AII type.
Experimental Eye Research 06/2014; 125. DOI:10.1016/j.exer.2014.06.019 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this pilot study was to test the effectiveness of a light therapy device (the Luminette®) on the delayed sleep phase syndrome (DSPS) in a group of adolescents (n = 10) between 15 and 18 years old (three girls and seven boys, average age 16.3 years old) affected by this syndrome. This study was conducted using an experimental single blind placebo-controlled design. The diagnosis of the DSPS among participants was established based on the criteria specified in the International Classification of Sleep Disorders – Second Edition (ICSD-2). The data were collected using two questionnaires: (1) Teen Sleep Diary (TSD) and (2) the Pediatric Daytime Sleepiness Scale (PDSS). The results indicated significant improvements in the experimental group (users of the real Luminette®) compared to the control group (users of the placebo Luminette®) with respect to the delay of sleep onset, the quality and the daytime sleepiness. This study underlines the importance of conducting further research on the Luminette® with a larger sample of adolescents with DSPS.
Médecine du Sommeil 04/2014; 11(2). DOI:10.1016/j.msom.2014.03.003
[Show abstract][Hide abstract] ABSTRACT: Purpose:
To examine the pattern of cone degeneration in the retina of a transgenic mouse model of Stargartd-like dystrophy (STGD3).
Investigations were performed on ELOVL4/TG1-2 transgenic (TG) mice and wild-type (WT) littermates from 1 to 24 months of age. Phenotypes were assessed by fundus imaging, fatty acid analysis, and electroretinogram (ERG) recording. Cone degeneration pattern was determined on retina whole mounts using immunohistochemistry and Voronoi domain analyses.
Consistent with low transgene expression, photoreceptors degenerate very slowly. At 1 month, anatomical structure and fatty acid composition of the TG retina is comparable with WT. Rod loss appears at 2 months, exhibiting a central to peripheral gradient, and fundus defects are observed at 3 months. In contrast, cone morphology, distribution and function are still normal at 12 months. Cone loss becomes apparent at 15 months when the outer nuclear layer is reduced to 3 to 4 photoreceptor rows. This process starts at the center of the retina and affects cone subtypes similarly. Very few cones remain at 24 months, after all rods have disappeared (18 months). Quantitative studies focusing on cones expressing M-opsin show a net increase in Voronoi domains and a significant decrease in regularity indexes only beyond 15 months.
Photoreceptor degeneration in this STGD3 mouse model follows the time course of a slow rod-cone dystrophy. The cone mosaic is preserved for almost 1 year after the onset of rod loss. This long delay provides an opportunity to examine rod-cone interactions during retinal degeneration and to test therapeutic effectiveness at protracting cone dysfunction.
[Show abstract][Hide abstract] ABSTRACT: Purpose The P23H rhodopsin mutation is an extensively studied model of ADRP. We evaluated the anatomical changes using SD-OCT and correlate the findings and retinal thickness values with immunocytochemistry. Functional changes were analyzed Methods Heterozygous P23H pigmented transgenic rats aged from P18 to P180 were studied. LE rats bred with Sprague Dawley (SD) 1 month old served as wild type controls. Visual acuity and contrast sensitivity evaluation was performed every month. Corneal ERGs were recorded under scotopic and photopic conditions. Retinal thicknesses at different levels (total thickness, ONL + RPE, ONL and IPL), fundus autofluorescence (FAF) and fluorescein angiography was performed in 3 animals at P150 using Spectralis OCT and HRA (Heidelberg Engineering, Germany). Retinas were immunostained for ICC. Results Retinal thicknesses diminution was seen in OCT sections, with a clear loss of ONL and morphological modifications. Statically differences were found between groups in all evaluated thicknesses. In the P23H rats, change in FAF was noted comparing to control group, as sparse autofluorescent dots. No relevant changes were observed in the angiography pattern. ICC showed a progressive decrease in ONL thickness. Functional changes were progressive with time. Conclusion Anatomical changes in pigmented P23H can be observed using SD-OCT and immunocytochemistry, with a good correlation between their values. SD-OCT and FAF are important tools for research in retinal degenerations.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
To assess the impact of early (dry) and late (wet/neovascular and/or atrophic) forms of AMD on panretinal function.
Light- and dark-adapted full-field ERG recordings were obtained over a 5-log-unit intensity range from both eyes of 25 patients with unilateral wet AMD. Fellow eyes showed various signs of dry AMD ranging from multiple medium-sized drusen to noncentral geographic atrophy. The leading edges of rod-isolated ERG a-waves were fitted to a quantitative model of phototransduction. ERG a- and b-wave amplitudes and implicit times were compared between wet and dry AMD eyes and from non-AMD eyes of age-matched subjects. A quantitative and objective assessment of dark adaptation was achieved by recording the recovery of the pure rod b-wave (postsynaptic depolarization of rod bipolar cells); b-wave amplitudes were measured at 120-second intervals for 20 minutes and normalized to the amplitude recorded at t = 20 minutes.
Delays in mixed a- and b-wave implicit times were recorded in both wet and dry AMD eyes. Time required to reach 50% of fully recovered responses was delayed in all wet and dry AMD eyes independently of dry AMD severity in the fellow eye. Generalized cone dysfunction and slower activation of the rod phototransduction cascade was noted in a subgroup of patients with advanced features of dry AMD in the fellow eye.
Patients with unilateral wet AMD display rod dysfunction in both their wet and dry AMD eyes. A subset of these patients display, in addition, bilateral cone dysfunction and delayed rod phototransduction activation, which may either reflect extensive morphologic change in advanced stages of AMD and/or represent a distinct phenotypic manifestation within the heterogeneous context of AMD as a disease.
[Show abstract][Hide abstract] ABSTRACT: Intra-uterine growth restriction (IUGR) has been associated with increased predisposition to age-related complications. We tested the hypothesis that rat offspring models of IUGR would exhibit exacerbated, age-related retinal dysfunction.
Female Sprague-Dawley rats (maintained at 11.5% O2 from gestational day 15 to 21 to induce IUGR) and control offspring (maintained at 21% O2 throughout pregnancy) had retinal function assessed at 2 months (young) and 14 months of age (aged) with electroretinogram (ERG) recordings. Retinal anatomy was assessed by immunofluorescence.
Deficits in rod-driven retina function were observed in aged IUGR offspring, as evidenced by reduced amplitudes of dark-adapted mixed a-wave Vmax (by 49.3%, P<0.01), b-wave Vmax (by 42.1%, P<0.001) and dark-adapted peak oscillatory potentials (by 42.3%, P<0.01). In contrast to the rod-driven defects specific to aged IUGR offspring, light adapted ERG recordings revealed cone defects in young animals, that were stationary until old age. At 2 months, IUGR offspring had amplitude reductions for both b-wave (Vmax by 46%, P<0.01) and peak oscillatory potential (Vmax by 38%, P<0.05). Finally, defects in cone-driven responses were further confirmed by reduced maximal photopic flicker amplitudes at 2 (by 42%, P<0.001) and 14 months (by 34%, P = 0.06) and critical flicker fusion frequencies at 14 months (Control: 42±1 Hz, IUGR: 35±2 Hz, P<0.05). These functional changes were not paralleled by anatomical losses in IUGR offspring retinas.
These data support that the developing retina is sensitive to stressors, and that pathways governing cone- and rod-driven function differ in their susceptibilities. In the case of prenatal hypoxia, cone- and rod-driven dysfunction manifest at young and old ages, respectively. We must, therefore, take into account the specific impact that fetal programming might exert on age-related retinal dystrophies when considering related diagnoses and therapeutic applications.
PLoS ONE 04/2013; 8(4):e61861. DOI:10.1371/journal.pone.0061861 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retinal dystrophies are predominantly caused by mutations affecting the visual phototransduction system and cilia, with few genes identified that function to maintain photoreceptor survival. We reasoned that growth factors involved with early embryonic retinal development would represent excellent candidates for such diseases. Here we show that mutations in the TGF-β ligand Growth Differentiation Factor 6, which specifies the dorso-ventral retinal axis, contribute to Leber congenital amaurosis. Furthermore, deficiency of gdf6 results in photoreceptor degeneration, so demonstrating a connection between Gdf6 signaling and photoreceptor survival. In addition, in both murine and zebrafish mutant models, we observe retinal apoptosis, a characteristic feature of human retinal dystrophies. Treatment of gdf6-deficient zebrafish embryos with a novel aminopropyl carbazole, P7C3, rescued the retinal apoptosis without evidence of toxicity. These findings implicate for the first time perturbed TGF-β signaling in the genesis of retinal dystrophies, support study of related morphogenetic genes for comparable roles in retinal disease and may offer additional therapeutic opportunities for genetically heterogeneous disorders presently only treatable with gene therapy.
Human Molecular Genetics 01/2013; 22(7). DOI:10.1093/hmg/dds560 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multiple Sclerosis is associated with a high incidence of depression, cognitive impairments and neuropathic pain. Previously, we demonstrated that tactile allodynia is present at disease onset in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). We have now monitored changes in object recogntion in mice with EAE to determine if altered nociceptive sensitivity is also associated with behavioural signs indicative of cognitive impairment in this model. At the onset of clinical signs, mice with EAE showed impairments in the novel object recognition (NOR) assay, indicative of deficits in cognitive functioning early in the disease course. At the spinal level, we found increased gene expression for the cytokines IL-1β, IL-6 and the glutamate transporter EAAT-2 that coicinde with increased nociceptive sensitivity and deficits in object recognition. Increased levels of EAAT-2 mRNA appear to be a response to perturbed proteins levels of the transporter as we found a loss of EAAT-2 protein levels in the spinal cord of EAE mice. To determine if changes in the levels of EAAT-2 were responsible for the observed changes in nociceptive sensitivity and cognitive deficits, we treated EAE mice with the β-lactam antibiotic ceftriaxone, an agent know to increase glutamate transporter levels in vivo. Ceftriaxone prevented tactile hypersensitivity and normalized performance in the NOR assay in EAE mice. These findings highlight the important interrelationship between pain and cognitive function in the disease and suggest that targeting spinally mediated pain hypersensitity is a novel therapeutic avenue to treat impairments in other higher order cortical processes.
[Show abstract][Hide abstract] ABSTRACT: Interneuronal subtype diversity lies at the heart of the distinct molecular properties and synaptic connections that shape the formation of the neuronal circuits that are necessary for the complex spatial and temporal processing of sensory information. Here, we investigate the role of Irx6, a member of the Iroquois homeodomain transcription factor family, in regulating the development of retinal bipolar interneurons. Using a knock-in reporter approach, we show that, in the mouse retina, Irx6 is expressed in type 2 and 3a OFF bipolar interneurons and is required for the expression of cell type-specific markers in these cells, likely through direct transcriptional regulation. In Irx6 mutant mice, presumptive type 3a bipolar cells exhibit an expansion of their axonal projection domain to the entire OFF region of the inner plexiform layer, and adopt molecular features of both type 2 and 3a bipolar cells, highlighted by the ectopic upregulation of neurokinin 3 receptor (Nk3r) and Vsx1. These findings reveal Irx6 as a key regulator of type 3a bipolar cell identity that prevents these cells from adopting characteristic features of type 2 bipolar cells. Analysis of the Irx6;Vsx1 double null retina suggests that the terminal differentiation of type 2 bipolar cells is dependent on the combined expression of the transcription factors Irx6 and Vsx1, but also points to the existence of Irx6;Vsx1-independent mechanisms in regulating OFF bipolar subtype-specific gene expression. This work provides insight into the generation of neuronal subtypes by revealing a mechanism in which opposing, yet interdependent, transcription factors regulate subtype identity.
Development 12/2012; 139(24):4644-55. DOI:10.1242/dev.081729 · 6.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: While some studies on dietary supplementation with docosahexaenoic acid (DHA, 22:6n-3) have reported a beneficial effect on memory as a function of age, others have failed to find any effect. To clarify this issue, we sought to determine whether supplementing mice with a DHA-enriched diet could alter the ability of synapses to undergo activity-dependent changes in the hippocampus, a brain structure involved in forming new spatial memories. We found that DHA was increased by 29% ± 5% (mean ± SE) in the hippocampus for the supplemented (DHA+) versus nonsupplemented (control) group (n = 5 mice per group; p < 0.05). Such DHA elevation was associated with enhanced synaptic transmission (p < 0.05) as assessed by application of a high-frequency electrical stimulation protocol (100 Hz stimulation, which induced transient (<2 h) increases in synaptic strength) to slices from DHA+ (n = 4 mice) hippocampi when compared with controls (n = 4 mice). Increased synaptic responses were evident 60 min poststimulation. These results suggest that dietary DHA supplementation facilitates synaptic plasticity following brief high-frequency stimulation. This increase in synaptic transmission might provide a physiological correlation for the improved spatial learning and memory observed following DHA supplementation.
[Show abstract][Hide abstract] ABSTRACT: With age, retina function progressively declines and A2E, a constituent of the toxin lipofuscin, accumulates in retinal pigment epithelial (RPE) cells. Both events are typically exacerbated in age-related retina diseases. We studied the effect of dietary docosahexaenoic acid (DHA, C22:6n-3) supplementation on these events, using a transgenic mouse model (mutant human ELOVL4; E4) displaying extensive age-related retina dysfunction and massive A2E accumulation.
Retina function was assessed with the electroretinogram (ERG) and A2E levels were measured in E4 and wildtype (WT) mice. Dietary DHA was manipulated from 1 to 3, 1 to 6, 6 to 12, and 12 to 18 months: 1% DHA over total fatty acids (E4+, WT+) or similar diet without DHA (E4-, WT-).
Increased omega-3/6 ratios (DHA/arachidonic acid) in E4+ and WT+ retinas were confirmed for the 1- to 3-month and 1- to 6-month trials. Although 1- to 3-month intervention had no effects, when prolonged to 1 to 6 months, RPE function (ERG c-wave) was preserved in E4+ and WT+. Intervention from 6 to 12 months led to maintained outer and inner retina function (ERG a- and b-wave, respectively) in E4+. At 12 to 18 months, a similar beneficial effect on retina function occurred in WT+; A2E levels were reduced in E4+ and WT+.
DHA supplementation was associated with: preserved retina function at mid-degenerative stages in E4 mice; prevention of age-related functional losses in WT mice; and reduced A2E levels in E4 and WT mice at the oldest age examined. These findings imply that dietary DHA could have broad preventative therapeutic applications (acting on pathologic and normal age-related ocular processes).
[Show abstract][Hide abstract] ABSTRACT: The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture.
In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam.
We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and identify Pten as an integral component of a novel cell positioning pathway in the retina.
PLoS ONE 03/2012; 7(3):e32795. DOI:10.1371/journal.pone.0032795 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate differentially expressed genes in eyecup and retina of the ELOVL4 transgenic mouse, a model of Stargardt-like macular dystrophy (STGD3).
We examined gene and protein expression in known pathways relevant to retinal degeneration using PCR arrays, Western blotting, and immunohistochemistry. Investigations were performed on ELOVL4 transgenic mice at 9 months, when 50% of rod (but no cone) photoreceptors had degenerated. Age-matched wild-type littermates served as controls.
Significant expression level changes were found in only 17 of the 252 genes examined. Nine were upregulated (Fgf2, Fgfr1, Ntf5, Cbln1, Ngfr, Ntrk1, Trp53, Tlr6, and Herpud1), and eight were downregulated (Ccl22, Ccr3, Il18rap, Nf1, Ccl11, Atf6β, Rpn1, and Serp1). Overexpression of FGF2 was detected at 1 month, before rod loss onset, and was maintained at high levels until cone loss (18 months). By 9 months, FGF2 overexpression was seen in photoreceptor cell bodies. Increased glial fibrillary acidic protein (GFAP) expression due to glial cell reactivity followed the same time course. Levels of NGFR/p75NTR remained invariant. Although present in rod outer segments at 1 month, the macrophage chemoattracting chemokine CCL22 became undetectable by 9 months, a likely consequence of progressive rod outer segment truncation.
At a mid-degeneration stage, major changes in gene expression in the ELOVL4 transgenic mouse retina included upregulation of Fgf2 and Fgfr1 and downregulation of Ccl22. Modulation of FGF2 occurred very early, concomitant with an increase in GFAP expression. Future studies will address which factors upstream of Fgf2 could provide potential therapeutic targets to slow photoreceptor degeneration in STGD3.