Nanoceria Decrease Vascular Permeability in a Mouse Model of AMD

Research Article

Austin Ophthalmol. 2016; 1(1): 1005.

Nanoceria Decrease Vascular Permeability in a Mouse Model of AMD

Cai X¹*, Seal S4 and McGinnis JF1,2,3*

¹Department of Ophthalmology, University of Oklahoma Health Sciences Center, USA

²Department of Cell Biology and Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, USA

³Department of Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, USA

4Advanced Materials Processing Analysis Center, University of Central Florida, USA

*Corresponding author: Xue Cai, Department of Ophthalmology, University of Oklahoma Health Sciences Center, 608 Stanton L. Young Blvd, Oklahoma City, OK 73104, USA

McGinnis JF, Department of Ophthalmology, University of Oklahoma Health Sciences Center, 608 Stanton L. Young Blvd, Oklahoma City, OK 73104, USA

Received: September 22, 2016; Accepted: October 25, 2016; Published: October 27, 2016

Abstract

The Very Low Density Lipoprotein Receptor Knockout (vldlr-/-) mouse is a model for a distinct form of Age-Related Macular Degeneration (AMD) called Retinal Angiomatous Proliferation (RAP). It is characterized by neovascularization, increased vascular permeability and retinal degeneration. We have reported that administration of nanoceria to pigmented vldlr-/- mice significantly inhibits the developmental neovascularization when injected at postnatal day (P) 7 and produces sustained regression of the existing neovascularization when applied at P28. In this study, we characterized the effects of the homozygous presence of the vldlr mutation on an albino background on retinal degeneration and neovascularization. We also examined the effects of increasing concentrations of nanoceria on: inhibition of the expression of Vascular Epithelium Growth Factor (VEGF), the number of retinal and choroidal neovascularization, the health of Retinal Pigment Epithelium (RPE) cells and the loss of RPE junctional proteins using albino vldlr-/- mice. Our data demonstrate that nanoceria function in a dose-dependent manner to up-regulate the expression of RPE65 and tight-junction proteins, down-regulate angiogenesis-stimulating factors, inhibit Blood-Retinal Barrier (BRB) breakdown and decrease vascular permeability in adult mice. These findings suggest that nanoceria are potential therapeutics for treatment of ocular diseases caused by RPE dystrophy and BRB dysfunction.

Keywords: Albino vldlr-/- mice; Nanoceria; RPE; Tight-junction proteins; Blood-retinal barrier

Abbreviations

AMD: Age-Related Macular Degeneration; BRB: Outer Blood- Retinal Barrier; CNV: Choroidal Neovascular “tufts”; FITC: Fluorescein Isothiocyanate; INL: Inner Nuclear Layer; Nanoceria: Cerium Oxide Nanoparticles; ONL: Outer Nuclear Layer; OPL: Outer Plexiform Layer; qRT-PCR: Quantitative Real Time RT-PCR; RAP: Retinal Angiomatous Proliferation; RNV: Retinal Neovascular “blebs”; ROS: Reactive Oxygen Species; RPE: Retinal Pigment Epithelium; TRX: Thioredoxin; VEGF: Vascular Epithelium Growth Factor; vldlr-/-: Very Low Density Lipoprotein Receptor Knockout; wt: Wild Type; ZO: Zonula Occludens

Introduction

The Retinal Pigment Epithelium (RPE) cells form a monolayer of cells in the back of the eye adjacent to the photoreceptors and play important physiological and functional roles in the process of conversion of light to neural signals [1,2]. The communication and cooperation between RPE cells and photoreceptors completes the visual cycle. The RPE cells, which contain pigmented granules, shield the photoreceptors from excessive light, and digest the aged outer segment discs of photoreceptors. The RPE cell also secretes growth factors, transports nutrients and provides iron channels [2]. It forms an outer Blood-Retinal Barrier (BRB) to control vascular fluid (as well as to absorb fluid from the retinal space) and exchanges nutrients and metabolic materials between the choroid and subretinal space [1]. This enables it to be highly selective in the modulation of the movement of oxygen and other molecules from the choroidal circulation system to the retina [3,4]. The tight junction complex forms the fundamental structure of the BRB and contributes to epithelial cell adhesion, communication and cellular movement. More than 40 proteins are found to be closely associated with tight junctions [5]. Among them, transmembrane claudins and occludin, and the scaffolding Zonula Occludens (ZO) proteins play essential roles in the formation and regulation of the BRB.

Breakdown of the BRB and consequences of the dysregulation of vascular permeability are closely associated with angiogenesis and can result in retinal edema [6-8]. This pathological condition is also correlated with an elevation of Vascular Epithelium Growth Factor (VEGF) [9,10]. The progression of the pathology of Age- Related Macular Degeneration (AMD) is predominantly and strongly correlated with oxidative stress-induced molecular and cellular injury, and the deposition of damaged proteins and other molecules in the RPE cells results in dystrophy of the RPE [11,12]. The central role of the RPE cell and the correlation of its dysfunction with the pathogenesis of AMD have been demonstrated by many laboratories [13,14]. The eventual degeneration of photoreceptors appears to be secondary to RPE senescence [15].

We have previously shown that catalytic inorganic Cerium Oxide Nanoparticles (nanoceria), which regeneratively scavenge Reactive Oxygen Species (ROS) and mimic the activities of the antioxidative enzymes, superoxide dismutase and catalase [16-18], have therapeutic effects against light-induced damage to the retina of albino Wild Type (wt) rats [19]. We have also shown that nanoceria prevent retinal degeneration in tubby mice [20,21] and inhibit the development of neovascularization and cause regression of the existing neovascularization in pigmented vldlr-/- mice [22,23]. Recently, we demonstrated that long-term retention of nanoceria in the retina does not induce any changes in retinal structure and function in albino rats [24], and we did not detect any increased inflammatory responses caused by any of the nanoceria concentrations tested in wt mice [25]. In the current study, we focus on the ability of nanoceria to provide protection to RPE cells, regulate tight-junctions and other junctional proteins, and to inhibit increased vascular permeability in adult albino vldlr-/- mice.

Materials and Methods

Intravitreal injection

Saline (1 μl), or saline with increasing concentrations of nanoceria from 0.001 mM (0.172 ng), 0.01 mM (1.72 ng), 0.1 mM (17.2 ng), 1 mM (172 ng) to 10 mM (1720 ng) were delivered into the vitreous of the albino vldlr-/- mice at P28 by injection as previously reported [23]. Uninjected vldlr-/- and wt Balb/C mice served as controls.

Vascular filling assay

The mice, at scheduled time points, were anesthetized and then 40 μl of 2.5% high molecular weight Fluorescein Isothiocyanate (FITC) - dextran (Sigma-Aldrich, FD-2000S) were injected into the left ventricle of the heart [22,23]. The anesthetized mice were killed 5 minutes later; the eyes were enucleated and fixed in 4% paraformaldehyde. The eyes were dissected, flat-mounted, observed and imaged as previously reported [22,23]. Eyes (20-30 per group) were analyzed and retinal neovascular “blebs” and choroidal neovascular “tufts” were counted using an Olympus MVX10 stereomicroscope. Data shown are mean ± SEM.

Fundus imaging and fluorescein angiography

Observation of the fundus and neovascularization were done as previously reported [23] with minor modification. Briefly, mice were anesthetized, the eyes were dilated, and the mice were placed on the bed of the Micron III system (Phoenix Research Labs, Pleasanton, CA). After the fundus was clearly seen and images taken, 20 μl of 5% AK-Fluor (Alcon) was intraperitoneally injected into the mouse. The photographs were captured 30 seconds, 60 seconds and 90 seconds after injection using StreamPix software and blue filters.

Optical Coherence Tomography (OCT)

Mice were fully anesthetized and eyes were dilated. One drop of refresh optive moisturizing solution was placed on the cornea. The mouse was put on the adjustable curset of the OCT machine (Bioptigen) and the head was held in a proper position, then the retina was scanned and images were saved.

Electroretinography (ERG)

Mice were dark adapted overnight, the eyes were dilated, and intensity scotopic ERGs were performed at P35 days with light intensities of 0.002, 0.02, 0.2, 2, 200 and 2000 cds /m2. Full field scotopic ERG with light intensity of 600 cds /m2 and photopic ERG with light intensity of 1000 cds /m2 were performed at P35 days (P35d), 3 months (P3m), and 7 months (P7m).

Immunocytochemistry and whole mount immunofluorescence staining

The eyes were collected, fixed, dissected as eyecups (SCR, Sclera-Choroid-RPE) without lens and cornea. For flat mount immunofluorescence staining, the dissected SCRs were blocked with 5% BSA, incubated with primary antibodies: either mouse anti-RPE65 (1:500, Millipore) or rabbit anti-ZO-1 (1:300, invitrogen) at 4°C overnight then incubated in anti-mouse or anti-rabbit AlexaFluor 488 for 1 hr at room temperature. After DAPI counterstaining, the SCRs were flat-mounted on the slides with RPE face-up and 4-6 radial cuts were made before coverslipping. For cryosectioning, the SCRs were embedded in OCT media and 10 μm sections were cut as previously reported [20,23]. The slide-mounted cryo-sections were blocked and incubated in the above primary antibodies at room temperature for 2 hrs, then incubated in anti-mouse or anti-rabbit AlexaFluor 488 for 1 hr at room temperature. After DAPI counterstaining the slides were coverslipped. Image capture was performed using a Nikon Eclipse 800 epi-fluorescence microscope.

Histology and quantitation of nuclei

The procedure for histology is the same as previously reported [20,23]. H & E stained slides from eyes at different developmental stages were observed and imaged with a Nikon Eclipse 800 microscope under 10x and 40x. For morphometry and quantitative histological analysis, three fields with 0.48 mm intervals between each field were imaged superiorly and inferiorly under 40x with the first image at a distance of 0.48 mm from ONH (Optic Nerve Head). The number of nuclei in the Outer Nuclear Layer (ONL) was determined (3-6 eyes per group) and the data shown are the averages of all measurements within the same age per group.

Quantitative Real Time RT-PCR (qRT-PCR)

Eyecups (3-5), without cornea and lens, from each group at P35 days, were collected and kept in TRIzol at -80°C. Total RNA isolation and cDNA synthesis are the same as previously reported [20]. For each sample, 20 ng of cDNA in triplicate was used for qRT-PCR reactions to determine the mRNA levels of VEGF. Primer sequences for the VEGF gene and the house-keeping gene (GAPDH) are the same as previously reported [23]. Relative expression levels were calculated [23] and are shown as mean ± SEM.

Western blot

Eyecups (3-8) from each group were collected. Protein extraction, quantitation, gel electrophoreses, membrane transfer and membrane development were the same as previously reported [20]. Soluble protein (50 μg) was loaded in each well of the gel. The following primary antibodies were used: sheep anti-VEGF and goat anti- Occludin (1:1000 and 1:750 respectively, Santa Cruz), mouse anti- RPE65 (1:2000, Millipore), anti-IL-1β (1:1000, Millipore), rabbit anti-ZO-1 (1:500, invitrogen), anti-TNF-a (1:1000, Millipore), and anti-IL-6 (1:1000, Proteintech). Rabbit anti-β-actin (HRP conjugate) (1:1000, cell signaling technology) or anti-GAPDH (1:2500, Abcam) antibody served as loading controls. The band detection and densitometric analysis of the bands were performed as previously reported [20,23].

Statistical analysis

One way ANOVA analysis with Bonferroni post hoc test and/or unpaired student t-test was performed and P value of less than 0.05 (P<0.05) was considered as a significant difference and is indicated in each figure.

Results

Retinal development and photoreceptor degeneration in albino vldlr-/- mice

Eyes at P7–365 were collected and processed for analysis of the development of the retina and neovascularization. There are no major differences in retinal morphology and the nuclear number in the ONL of albino vldlr-/- and wt Balb/C mice at P7 and P14 (Figure 1A) although the initiation of neovascularization in vldlr-/- mice occasionally appeared at P14. The obvious retinal structural changes, because of the penetration of neo-blood vessels from the Outer Plexiform Layer (OPL) through the ONL and connecting to the choroid, were always seen at P21 (Figure 1A). Histological analysis indicated that the mutant retinas at P28 have 91.39% of the nuclei present in age-matched wt littermates (Figure 1B). Severe ONL abnormalities with regional increases in RPE layers, accompanied by photoreceptor cell death (Figure 1A, Figure 1B), are frequently observed after P35. At P49, retinal detachment occurred because of drusen formation (Figure 1A), and at P70, about 20% of the photoreceptors were lost. Severe retinal degeneration occurred by P5m, when 37% of photoreceptor cells in the mutant retinas were absent (Figure 1B). Irregular retinal structures, such as a thickened RPE with multiple layers of cells, rosette-like structures in the ONL, thinning of the ONL and/or INL (Inner Nuclear Layer) beneath the lesion area, and the fusion of the retinal neovascular vessels and choroidal neovascular vessels represent the typical retinal morphology in vldlr-/- mice (Figure 1A). OCT images of the retina of living vldlr-/- mice at P35d, P3m and P7m revealed the progressive development of the lesions, the enlarged fused neovasculature, and retinal thickening indicative of edema, all of which eventually cause severe retinal detachment (Figure 1C). To further examine the retinal degeneration and its function in response to light, full field ERG (Figure 2A) was performed at P35d, P3m and P7m of age. The amplitude of cone ERG at P35d had decreased to 71.5% of wt and it further decreased to 61% of wt by P7m. In contrast, the rod function has no large changes at P35d when measured by full field ERG. However, intensity ERG (Figure 2B) at P35d demonstrated that the rod sensitivity to light in vldlr-/- mice is statistically lower than in wt mice. Rod response to the light in vldlr-/- mice declined to 76% of wt at P3m (a-wave only, the b-wave has minor changes) and it was only 69% of wt at P7m (Figure 2A). These data indicate that cone degeneration occurred earlier than rod degeneration and degeneration of secondary neurons occurs later than the primary neurons.