Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008

Fig. 1 BALB/c mice display significant losses in visual acuity following 24 hours of exposure to intermittent blue and intermittent white light (P <0.001). Included in the graph are the visual acuity of mice exposed to no bright light (white bar, n=51 trials, 6 mice), 24 hours of blue light (light grey bar, n=12 trials, 2 mice), 24 hours of intermittent blue light (dark grey bar, n=16 trials, 2 mice), 24 hours of intermittent white light (grey bar, n=17 trials, 2 mice). Statistics were calculated using one-way ANOVA with Newman-Keuls post hoc test. Bar represents mean ± standard error

than to intermittent blue light (P <0.01). 24 hours of continuous blue light did not significantly affect either measure. We did not detect changes in visual acuity or contrast sensitivity of C57BL/6J mice exposed to 7 days of continuous or intermittent blue or white light exposure (data not shown).

3.2 Retinal Function

Losses of retinal function assessed by ERG recordings correlate well with the deficits in visual function uncovered with optomotor behavioral testing. In BALB/c mice we detected a significant difference in the maximum ERG b-wave amplitudes

Table 1 Summary of acuity in BALB/c mice

Fig. 2 BALB/c mice display significant losses in retinal function following 24 hours of exposure to intermittent white light (P <0.004; unpaired, two-tailed t-test). b-wave intensity-response functions for control BALB/c mice (filled circles, n=3) and BALB/c littermates exposed to 24 hours of intermittent white light (open circles, n–2). Bar represents mean ± standard error

in mice treated with intermittent white light (172 ± 32 V, n=2) versus unexposed animals (845 ± 59μV, n=3) (P <0.004). The curve fitting the control mice data was divided by 4.9 to fit the exposed mice data (Fig. 2). Such scaling of the ERG intensity-response functions indicates that intermittent white light exposure altered the gain but not the sensitivity of the retinal responses. Conversely, we did not detect a difference in the C57BL/6J maximum b-wave amplitude following 7 days of continuous blue light, continuous white light, or intermittent white light (data not shown).

4 Discussion

BALB/c mice exhibit losses in visual function as assessed by optomotor behavior and retinal function as assessed by ERG in response to 24 hours of intermittent light exposure. In contrast, 24 hours of continuous blue light was only mildly effective in reducing retinal and visual function in BALB/c mice. At first, this result is perplexing, as mice treated with continuous light are exposed to a much higher greater total photon flux. To reconcile this result, one must consider the mechanism of extreme light damage. Intense intermittent white or blue light exposures provided six periods of complete rhodopsin bleaching in BALB/c mice based on previously published rhodopsin regeneration kinetics (Wenzel et al., 2001). Although continuous light provides a greater total photon flux, it most likely evokes only one complete rhodopsin bleaching event. Thus, in terms of effective “bleaching power” intermittent light is far more powerful and therefore more destructive. The data fully support this assertion, as the most effective exposure paradigm tested was that of intermittent light.

ERG intensity-response functions from control and intermittent light exposed BALB/c mice exhibit similar shapes, but vastly different amplitudes. In fact, the BALB/c treated function plotted in Fig. 2 is simply a scaled down version of the control curve (4.9 times smaller). This is consistent with the currently accepted mechanism of light-induced damage – photoreceptor apoptosis (Wenzel et al., 2005). Loss

of photoreceptors reduces the generation of the ERG leading to a simple scaling of ERG amplitudes, which is what we found (Fig. 2).

C57BL/6J mice did not experience losses of visual function or retinal function under any of the extreme light paradigms tested. Tests included 7 days of continuous blue light exposure, 7 days of continuous white light exposure and various intermittent white light exposure paradigms (data not shown). The RPE65 mutation in C57BL/6J mice certainly plays a role, but it may not explain the extremely low light damage susceptibility of these mice. B6;129S(N2) mice have the RPE65 variant, but have at least a ten times higher susceptibility to light damage (Wenzel et al., 2001). Extremely long intermittent light paradigms may lead to damage in C57BL/6J mice, but our test conditions did not.

We find that losses of visual function as assessed by optomotor behavior correlate with losses of retinal function measured with ERG. This may not necessarily be the case, as retinal function could be reduced without comparable loss of visual function. However, it would be much harder to explain losses of visual function without concurrent losses in retinal function if one is studying a retinal based disease. Thus far we have used behavioral testing to detect losses of vision in mildly hypoglycemic Gcgr-/- (Umino et al., 2006a), Isl-/- conditional knockout (Elshatory et al., 2007), and Gnat2cpfl3 mice (Alexander et al., 2007). The ability to test individual eyes and specific aspects of vision make optomotor behavior testing attractive for rapidly assaying effects of gene mutations or drug treatments. Behavioral testing of visual function can be performed rapidly and accurately throughout the duration of a study, minimally effecting exposure regimens. This behavioral technique holds great promise for testing the effects of extreme light on visual function.

Acknowledgments We would like to thank Dr. Daniel Organisciak (Wright State University; Dayton, OH) for loan of the light exposure chamber and for helpful suggestions and Rebekah Hafler (SUNY Upstate Medical University; Syracuse, NY) for technical assistance. This work was supported by grants from Fight for Sight (D.E), the National Eye Institute (EY00667) to R.B. and (1F32EY017246) to D.E., NASA, Research to Prevent Blindness, and the Lions of Central NY.

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ERG Responses and Microarray Analysis

of Gene Expression in a Multifactorial Murine Model of Age-Related Retinal Degeneration

Goldis Malek, Jeffery A. Jamison, Brian Mace, Patrick Sullivan, and Catherine Bowes Rickman

1 Introduction

Age-related macular degeneration (AMD) is a late-onset neurodegenerative retinal eye disease that manifests as progressive loss of central vision. It is a common disease caused by the interaction of genetic predisposition and exposure to modifiable risk factors. Risk factors identified to date include but are not limited to: advanced age, environmental factors (e.g. smoking, diet) (Chua et al. 2006; Guymer and Chong 2006; Seddon et al. 2006; Weale 2006) and genetics (e.g. complement factors H & B, LOC387715, and apolipoprotein E [APOE]) (Klaver et al. 1998; Souied et al. 1998; Schmidt et al. 2002; Edwards Iii et al. 2005; Hageman et al. 2005; Haines et al. 2005; Klein et al. 2005; Conley et al. 2006; Gold et al. 2006). We developed a murine model that closely approximates changes seen in human AMD, by combining advanced age, human APOE isoform and a high fat, cholesterol-rich (HF-C) diet, three risk factors associated with the human disease (Malek et al. 2005). We determined that aged APOE4 mice fed a HF-C diet develop characteristic lesions of AMD including retinal pigment epithelial (RPE)-pigmentary changes, thick lipid-rich diffuse and focal basal deposits and growth factor immunopositive neovascular lesions. These changes were not detected in any of the control, human APOE3 expressing mice regardless of diet consumed, nor were there any pathologies detected in young APOE4 animals.

The goal of the current study was to identify altered patterns of retinal and RPE/choroid gene expression in mice with ‘AMD’ pathology versus normal eyes, based on the hypothesis that these differences will specifically reflect multifactorial AMD-risk factor-induced retinal changes that could identify genes and cellular pathways involved in progression of disease. We also investigated retinal function changes, which were then correlated with morphological changes detected in the retinas of the affected animals.

G. Malek

Department of Ophthalmology, Duke University, Durham NC, USA, Tel: 919-684-0820, Fax: 919-684-3687

e-mail: gmalek@duke.edu

2 Gene Expression Profiling

To identify genes affecting the development of, or protection from, AMD-like pathologies in our animal model, microarray based gene expression profiles of retina/RPE/choroid were generated as follows: we used human APOE targeted replacement mouse lines, expressing either APOE3 or APOE4 that were created by replacing the coding sequences of mouse apoE with human APOE allele-specific coding sequences (Sullivan et al. 1997). Aged male mice (mean age 80–122 wks) of each genotype (n=3/genotype) were bred and housed conventionally, fed a HF-C diet (35% fat, 20% protein, 45% carbohydrates, 1.25% cholesterol, 0.5% sodium cholate) for 8 weeks or normal chow (4.5% fat, 0.02% cholesterol). Left eyes were prepared for histopathology as described previously (Malek et al. 2005). Right eyes were enucleated, fixed in RNAlater and stored at –20◦C until use. Total RNA was isolated from the retina/RPE/choroid using TriZol-glycogen or Qiagen RNeasy lipid tissue mini kit. Samples were biotin-labeled for hybridization following amplification when necessary. Expression analysis was performed using Operon Mouse Oligo Set, version 3.0 spotted array GeneChips, representing 30,000 genes. Samples were hybridized onto microarray slides and visualized with the GeneChip scanner. Data were normalized and clustered using GeneSpring GX software.

Two RNA based approaches were used to validate the differential gene expression profiles obtained; quantitative real-time PCR of selected genes and large-scale transcript profiling on a novel, high-density microarray profile from Illumina (San Diego, CA). A new cohort of n=6–8/genotype/diet samples (retina/RPE/choroid RNA), was used to probe Illumina mouse Sentrix Beadarrays. This resulted in identification of both a larger number of total and differentially expressed genes. A manuscript detailing the complete Illumina based profiles is in preparation.

3 AMD-Related Pathways

Comparative analysis of the gene expression profiles of APOE4 mice with AMD pathology versus controls confirmed differential expression of genes previously implicated in the pathogenesis of AMD including inflammatory genes, oxidative stressand lipid-related genes. Also identified as differentially expressed were extracellular matrix molecules (e.g. TIMP3, ADAM15, extracellular matrix protein 1, retinol binding protein, fibronectin), and apoptotic genes (e.g. vascular endothelial zinc finger, TNF receptor). Furthermore, retinal synaptic genes, including synapsin, synaptotagmin, and piccolo, which based on preliminary immunohistochemical investigation appear to be disorganized in regions above focal and diffuse sub-RPE deposits in aged, APOE4 mice fed a HF-C diet, were also differentially expressed (Data not shown). These finding support the hypothesis that these ‘diseased’ mice share common pathogenic mechanisms with human AMD and provide further insight into the still unknown cholesterol and lipid induced changes in the eye and

Table 1 Candidate genes were prioritized and clustered based on pathways and processes implicated in AMD pathogenesis. Fold change is the statistically significant differential expression between APOE4 HF-C and normal diet samples, p<0.05

their involvement in AMD. Select differentially expressed genes identified on the Operon microarrays and their AMD-associated pathways are shown in Table 1.

4 Retinal Changes

4.1 Electroretinogram (ERG) Recordings

The ERG recordings were obtained from animals, dark adapted for at least 12 hours. Each animal was anesthetized with a ketamine/xylazine cocktail, pupils were dilated and after the animal stabilized on a 37◦C warming pad, ERG tracings were recorded using a silver wire test electrode placed in contact with the eye along with a drop of 2.5% hydroxypropyl methylcellulose. Mice were placed in a photopic stimulator chamber where the animal was exposed to flashes of light (max intensity of 1000 cd-s/m2 attenuate in 1 log steps, starting from 0.0005). The a-wave amplitude was measured from baseline to the a-wave trough, and the b-wave amplitude was measured from the a-wave trough to the b-wave peak.

There was a significant reduction in a- and b-wave amplitude in APOE4 HF-C mice versus mice on the normal diet (Fig. 1). No significant change was seen in the implicit times of the a-waves between the groups. There was a slight increase in the b-wave implicit timing of the APOE4 HF-C, though this was not statistically significant. Within the APOE4 HF-C cohort, ERG recordings fell broadly into three groups based on their average a- and b- wave amplitudes; high, med, and low. The degree of histopathological changes observed in affected aged, HF-C fed APOE4s varied in each animal in a manner reminiscent of pathologies documented in human

Fig. 1 Graph of intensity of a- and b-waves (A) and sample ERGs (B) from APOE4 mice aged over 65 weeks and fed a normal or high fat, cholesterol-rich (HF-C) diet

AMD eyes (Malek et al. 2005). Therefore, we wondered whether the heterogeneity in ERG recordings in affected animals would correlate with the morphological changes.

4.2 Morphological Changes

Pathologies in aged APOE4 mice fed and HF-C diet ranged in severity with some animals developing choroidal and retinal neovascularization ( 10%), some developing focal and diffuse sub-RPE deposits ( 75%) and some maintaining overall

Fig. 2 Light micrographs from one micron plastic sections of posterior retinas of aged APOE4 expressing mice fed a high fat and cholesterol-rich diet, stained with toludine blue. Retinal pigment epithelium (RPE) contains vacuoles (A). Presence of ‘drusen-like’ deposits located between the RPE and Bruch’s membrane (B; asterisks). Neovascularization confined to a sub-RPE region (C). Note, decreased thickness of the outer nuclear layer (ONL) overlying deposit and neovascularization in panels B and C. Magnification 40X. INL=inner nuclear layer, OPL=outer plexiform layer, IS=inner segments of photoreceptors, OS=outer segments of photoreceptors

Table 2 Measurements of retinal layer thickness and cell counts in aged APOE4 mice fed and high fat cholesterol-rich diet correlated to ERG a- and b-wave amplitude groups

normal retinal morphology ( 10%, Fig. 2). Retinal morphometric analysis was performed on plastic embedded tissue cross sections that bisected the optic nerve. Retinal layers were measured and cell numbers were counted in an area adjacent to the optic nerve head and in an area in the far periphery. A total of 10–12 sections were measured and counted and the results were averaged. Local or diffuse sub-RPE deposits were seen in approximately 76% of the mice, belonging to all three functional groups. Based on measurements of thickness of retinal layers and number of cells, there were no significant differences between ERG groups (Table 2). However, in regions immediately adjacent to deposits or neovascularization, some thinning of the outer nuclear layer as well as shortening of the photoreceptor inner and outer segments were documented (Fig. 2B, C).

5 Conclusions

ERG recordings of APOE4 HF-C mice demonstrate statistically significant decreased a- and b-wave amplitudes. Though gross morphological differences were not seen in the retinal layers, gene expression profiles obtained by microarray analyses suggest that changes may be occurring at a transcriptional and potentially translational level, which warrants further investigation. Several synaptic markers were differentially expressed in the APOE4 HF-C microarray samples including synaptotagmin, synaptophysin and piccolo. Similar changes have recently been demonstrated in human AMD eyes using these markers (Johnson et al. 2003; Johnson et al. 2005). Changes in synaptic terminals including synaptic retraction occurred in regions overlying drusen in human eyes. Preliminary immunolocalization experiments indicate that this is the case for APOE4 HF-C mice as well (manuscript in preparation).

Acknowledgments The authors gratefully acknowledge scientific input from Drs. Linc Johnson, Patrick Johnson, and Monte Radeke, technical assistance from Jessica N. Ebright and Peter Saloupis and the following funding agencies: AHAF MDR (CBR), NEI P30 EY005722, A Ruth and Milton Steinbach Award (CBR), Alcon Basic Research Funds (CBR) and Research to Prevent Blindness Core grant.