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

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Nanoceria Particles Prevent ROI-Induced

Blindness

Junping Chen, Swanand Patil, Sudipta Seal, and James F. McGinnis

1 Introduction

Retinal degeneration caused blindness, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), retinitis pigmentosa (RP) and retinal detachment, is a major problem in clinical ophthalmology. Although genetic modifications are responsible for most retinal degenerative diseases, there is increasing evidence showing that reactive oxygen intermediates (ROIs), the byproducts of the oxidative metabolic reactions, are closely involved in the process of photoreceptor cell degeneration (Beatty et al., 2000; Maeda et al., 2005; Wenzel et al., 2005). These ROIs, including hydrogen peroxide, hypochlorite ions, hydroxyl radicals, hydroxyl ions and superoxide anions (Beatty et al., 2000), react with almost any nearby DNA, RNA, lipid, carbohydrate or protein. They are produced primarily by the normal oxidative metabolism that occurs in the mitochondrial respiratory chain. Photoreceptor cells are extremely sensitive to ROI-induced damage. This is not only because they are continuously exposed to the deleterious effects induced by photons of light, but also they have the highest rate of oxygen metabolism of any cells in the body (Yu and Cringle, 2005) and their outer segments contain high level of polyunsaturated fatty acids. The high consumption of oxygen results in the production of a large amount of ROIs. In consequence, ROIs result in the cytotoxic effect referred to as oxidative stress, which has been implicated as one of the initial causes of numerous eye diseases. It has been shown that, in the retinal degenerative diseases, irrespective of the initiating defect, the intracellular concentration of ROIs rises chronically or acutely and activates a cell death pathway (Lewis et al., 1991; Caldwell et al., 2003; Emerit et al., 2004).

Several therapeutic strategies have been tested to treat retinal degeneration in animal models by reducing ROIs (Wenzel et al., 2005). We are introducing a new material, vacancy engineered nanoceria particles, and testing its ability to

J.F. McGinnis

Oklahoma Center for Neuroscience, Dean A. McGee Eye Institute, Department of Department of Cell Biology, Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA, Tel: 405-271-3695, Fax: 405-271-3721,

e-mail: james-mcginnis@ouhsc.edu

reduce ROIs (Chen, 2006). Cerium is a rare earth element in the lanthanide series. Cerium oxide (CeO2) is the oxidized form of the element. Cerium has both +3 and +4 states and may flip-flop between the two in a redox reaction (Suzuki et al, 2001; Herman, 1999; Conesa, 1995), during which oxygen vacancies or defects are formed in the lattice structure by loss of oxygen and/or its electrons. The nanocrystalized CeO2 molecules not only retain oxygen vacancies in the crystal structure (Patil et al., 2006), but they also are about 5nm in diameter which allows for easier passage through cell membranes. Thus we hypothesized that nanoceria particles, owing to their chemical and physical structure, can protect photoreceptor cells from lightinduced ROI-mediated damage.

2 Methods

2.1 Primary Culture of Retinal Neurons

Primary dissociated cell cultures of rat retinas were established from 0- to 2-day-old rat pups as described (McGinnis et al., 1999).

2.2 Measurement of Intracellular ROI

Intracellular ROI production was measured by flow cytometry using 29,79dichlorofluorescein diacetate (DCFH-DA), an oxidant-sensitive fluorescent probe. In the presence of intracellular peroxides, H2DCF is oxidized to a highly fluorescent compound, 2,7-dichlorofluorescein (LeBel et al., 1992). The retinal cells were exposed to nanoceria particles for 12h, and then incubated with 10mM DCFH-DA at 37◦C for 30 min after washes. The cells were then incubated with 1mM H2O2 at 37◦C for 30 min after the excess DCFH-DA was washed off with phosphate buffer saline. The cells were then harvested with trypsin. The intensity of fluorescence was detected by flow cytometry with an excitation filter of 485 nm. The ROI level was calculated as a ratio of mean intensity of experimental cells/mean intensity of control cells.

2.3Intravitreal Injection and Light-induced Photoreceptor Degeneration

Rats were anesthetized, pupils dilated, a topical anesthetic applied to the cornea, and 2 μl of 0.1, 0.3 or 1 μM nanoceria particles in 0.9% NaCl were injected. Controls were injected with 2 μl of 0.9% NaCl. For light-induced photoreceptor degeneration, the rats were exposed to 2,700 lux (measured with a photometer) of constant light for 6 h. During light exposure, rats were maintained in transparent polycarbonate cages (one or two rats per cage) with stainless-steel wire covers. A water bottle

was kept at the side of the cage and food was placed in the bottom of the cage on the bedding. They were returned to cyclic light for 7 days before the experiment was ended.

2.4Functional Rescue of Photoreceptor Cells as Evaluated by Electroretinography

The ERG experiments were performed as described previously (Cao et al., 2001). The animals used in each set of experiments were all the same sex (males) and were taken from the same litter. They were maintained in the same room and treated identically. Animals were kept in total darkness overnight before ERGs were recorded. For quantitative analysis, the A-wave amplitude was measured as the difference between baseline and the peak of the A-wave, and the B-wave amplitude was measured as the difference between the peaks of the A- and B-waves.

2.5 Morphologic Evaluation by Quantitative Histology

After ERG recordings, the rats were killed by an overdose of carbon dioxide, the eyes enucleated, fixed in Perfix, embedded in paraffin, and 5-μm-thick sections were cut along the vertical meridian so that the superior and inferior hemispheres were separated by the optic nerve. To evaluate quantitatively the morphologic changes using H&E stained sections, we measured the ONL thickness at 220-μm intervals, starting at the optic nerve head and moving along the vertical meridian toward the superior or inferior ora serrata. The mean ONL thickness of each point was then calculated from the retinas of at least four eyes.

3 Results

3.1Nanoceria Particles Inhibit Intracellular Accumulation of ROIs.

To study the ability of nanoceria particles scavenging intracellular ROIs, we used the intracellular ROIs maker, DCFH-DA. We found that the nanoceria particles prevent the intracellular accumulation of ROIs in cultured retinal neurons to which H2O2 was added. The data are shown in Fig. 1 and demonstrate that the nanoceria particles, even at 5 nM, when present for 12 h, are effective in inhibiting the H2O2- induced rise of intracellular ROIs.

Fig. 1 Inhibition of ROIs by nanoceria particles. Pretreatment of cultured retinal neurons with nanoceria particles (1, 3, 5, 10 and 20 nM) for 12 h inhibits the intracellular accumulation of ROIs in response to exposure to 1 mM H2O2 for 30 min. The protection is dose-dependent. (Statistical analysis was done by one-way ANOVA and Newman – Keuls test for post hoc analysis. Data are shown as mean ±s.d., n=3, P < 0.05)

3.2in vivo Protection of Photoreceptor Cells Morphology and Function

The cell culture data prompted us to test the efficacy of the nanoceria particles in vivo. We used a light-damage animal model in which albino rats are intravitreally injected with 2 μl of saline or a 0.1, 0.3 or 1.0 μM suspension of the nanoceria particles in saline and three days later were exposed to 2,700 lux of light for 6 h. The retinal function is evaluated by ERGs which were conducted 7 days after exposure. The quantification of ERG A- and B-wave amplitudes (Fig. 2A) clearly demonstrates the functional improvement of retinas following treatment with nanoceria particles from 0.1 to 1.0 μM. This is especially evident in the eyes pretreated with 1 μM nanoceria particles, which had 79% and 87% of the control A- and B-wave amplitudes, compared with 22% and 26% for the retinas with 0.9% NaCl vehicle treatment.

It is known that the superior retina is more sensitive to light damage than the inferior retina. The averages of the ONL thickness of the superior region are showed in Fig. 2B and demonstrate that the nanoceria particles are effective in protecting the photoreceptor cells in that region. Injections of 0.1 μM and 0.3 μM suspensions are partially protective, but the data obtained for a 1.0 μM suspension demonstrate almost complete protection and are essentially indistinguishable from those of the control animal, which was not exposed to light.

Fig. 2 Nanoceria particles provide pan-retinal protection against light damage. (A) Summation of the ONL thickness across the superior hemisphere provides a quantitative assessment of the protective effects of the nanoceria particles in the most sensitive hemisphere. (B) Mean of the ERG B-wave and A-wave amplitudes are shown for each group of animals. Statistical analysis was done by one-way ANOVA and Newman – Keuls test for post hoc analysis. The results are expressed as mean amplitude ±s.d. (n=6 for each point). Control animals had no injections and were not exposed to bright light. LE animals had no injections and were exposed to bright light

3.3Nanoceria Particles can Rescue Retina Function After Light Damage

To determine if the nanoceria particles had any ability to rescue photoreceptor cells after they had been exposed to damaging light, rats were subjected to 6 h of 2,700 lux light and were injected intravitreally, 2 h later, with 2 μl of a 1 μM nanoceria particle suspension. The retinal function of the animals was determined seven days later using ERG. The summary of the ERG data (Fig. 3) demonstrates that a significant amount of retinal function is rescued by post-treatment with the nanoceria particles.

4 Discussion

In this report, we for the first time showed that the vacancy engineered particles have the ability to scavenge intracellular ROI, to abolish the early events in the degeneration process and to prevent photoreceptor cell degeneration induced by light. We do not know how the nanoceria particles are taken up into photoreceptor cells, nor the process by which they are eliminated. However, because ROIs are formed within the photoreceptor cells in response to light damage, and because ROIs react over very short distances, we think that the nanoceria particles enter the photoreceptor cells. Because our data demonstrat that the nanoceria particles prevent the peroxide induced increase in the intracellular concentration of ROIs in cul-

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An in-vivo Assay to Identify Compounds Protective Against Light Induced Apoptosis

Yogita Kanan, Anne Kasus Jacobi, Kjell Sawyer, David S. Mannel, Joyce Tombran Tink, and Muayyad R. Al-Ubaidi

1 Introduction

Age-related macular degeneration (AMD) is the major cause of blindness in the elderly. Vision loss is caused by the death of cone photoreceptor cells in the macula. Approximately 1.65 million Americans over the age of 50 have the disease (Prevent Blindness America). Very little is known about the causes and mechanisms of photoreceptor death in this condition.

We are interested in understanding AMD and finding drugs that will alleviate this condition. In our laboratory, we have developed a cell line (661W) from a retinal tumor in mice expressing SV-40 T antigen in the photoreceptor cells (Al-Ubaidi et al., 1992). The cell line expresses cone specific antigens and therefore it is a valuable tool to study macular degeneration (Tan et al., 2004). Since light is considered to be a contributing factor in macular degeneration (Tomany et al., 2004), we designed an assay where we caused cell death in 661W by light stress in the presence of the chromophore (Kanan et al., 2007). These cells do not contain an endogenous chromophore but our previous results showed that preincubation of the cells with 10 μM 9-cis retinal or all-trans retinal potentiates the light damage, whereas all- trans retinol at the same concentration does not. Light causes isomerization of 9-cis retinal to all-trans retinal, making it the likely candidate that mediates light damage. Examples of the utilization of this assay to identify protective compounds against cell death are presented here. Two drugs, pigment epithelium derived factor (PEDF) and decosahexanoic acid (DHA), are tested for their protective effects against light damage.

We then tested three retinol dehydrogenases (RDH) 8, 11, and 12, upon transfection into 661W cells, for their ability to protect the cells from light induced cell death. Retinol dehydrogenases detoxify all-trans retinal by converting it to all-trans retinol.

Y. Kanan

Department of Cell Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd. (BMSB781), Oklahoma City, OK 73104, USA Tel: 405-271-2408

e-mail: ykanan@ouhsc.edu

2 Methods

2.1 Growth of Cells for Light-Induced Apoptosis Experiments

Twenty thousand cells were added in each well of a 96-well tissue culture plate and allowed to grow overnight. The next morning, the medium was replaced with a 100-μl aliquot of fresh medium containing 10 μM 9-cis retinal or 9-cis retinal and the protective drug in the dark, and the cells were returned to the incubator for 4 hours to allow for the uptake of retinoids. After 4 hours, the media was again removed and replaced with media containing the protective drug and then exposed to light at 30,000 lux for 4 hours at room temperature. Cells kept in the dark served as dark controls. Cells replaced with media containing drug were compared to cells replaced with media alone to test the protective effects of the drug. In the case of cells transfected with retinol dehydrogenase genes, we compared these cells with cells that were not transfected to assess protection.

2.2Expression Plasmids, Transfection and Selection of Stable Clones

RDH8-Flag, RDH11-Flag, and RDH12-Flag encoding epitope-tagged versions of these mouse RDHs were generated by PCR and cloned into the pTarget vector (Promega, Madison, WI). All expression plasmids were verified by DNA sequencing of the entire coding regions and cloning sites. 661W cells were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. To select for stable transfectants, 661W cells were grown in G418-containing medium (1 mg/ml) for 3 weeks. G418-resistant colonies were collected and clonal cell lines were obtained by serial dilution in 96-well plates and assayed for Flag-tagged RDH expression by immunoblot analysis with anti-Flag antibody. Once established, stable cell lines were maintained in G418-containing medium.

2.3 Viability Assay

Viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) according to the paper (Kanan et al., 2007). Data were graphed with commercial software (Prism; GraphPad Software). The graph was plotted by plotting the viability percent of the cells surviving compared to their dark control. The percentage of viable cells was calculated by averaging the ratios of absorbance readings of cells in the light to the dark control cells, assuming that the dark control cells were 100% viable, and the average percentage was determined.

Each experiment was performed at least three times, with 12 replicates for each treatment. Cells that underwent light treatment were compared with dark control