¡¡¡¡ARPEª²19 Cells Culture ARPEª²19 cell line was purchased from ATCC Company (American Type Culture ollection, Manassas, VA). It was incubated in 37¡æ, 50mL/L CO2 condition. Growth medium was composed of 1¡Ã1 mixture of Dulbecco¬ðs modified Eagle¬ðs medium (DMEM) and Ham¬ðs F12 medium containing 1.2g/L sodium bicarbonate, 2.5mmol/L Lª²glutamine, 15mmol/L HEPES, 0.5mmol/L sodium pyruvate and 100g/L fetal bovine serum (All purchases from Invitrogen Corporation, Carlsbad, CA). Confluent cultures were released by digestion with 2.5g/L trypsinª²0.2g/L EDTA (Sigmaª²Aldrich, St. Louis, MO).

¡¡¡¡Cell Proliferation Assay ARPEª²19 cells were grown in 96ª²well tissue culture plates overnight. Medium was then replaced by fresh medium or various concentrations of NaIO3 (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100mg/L). After incubation for 48 hours, cells were washed with Dulbecco¬ðs phosphateª²buffered saline one time and further incubated with 100g/L 3ª²(4,5ª²dimethythiazolª²2ª²yl)ª²2,5diphenyl tetrazolium bromide (MTT) 100¦ÌL for exactly 4 hours. Media was then removed by aspiration, 100¦ÌL DMSO was added into each well and dishes were shaken for 2 minutes to dissolve cells. Light absorbency in each well was read at 570nm using a Spectra Count plate reader (Packard BioScience, Meridan, CT). Proliferation rate was valued by NaIO3 treated cells comparing to normal cells. So the proliferation rate of normal cells was assigned as 100%.

¡¡¡¡Statistical Analysis Both eyes of each animal were used in the experiments. Cell culture was repeated 6 times and 6 wells were used each time for one group. Student t test was used for statistical analysis. Oneª²tail tª²test was used for in vivo experiª²ment and twoª²tail tª²test was used for in vitro part.

¡¡¡¡RESULTS

¡¡¡¡ERG Recordings After ACª²ERG recording, we measured aª²wave from baseline to the first negative trough; maximum bª²wave amplitude (Vbmax) from the first negative trough (aª²wave) to the first positive peak of the bª² wave (Figure 1A). For the DCª²ERG recording (Figure 1B), the second positive peak followed bª²wave was cª²wave. The amplitude of which was measured from the trough after bª²wave (which was after potential, AP) to the peak of cª²wave. The amplitude of FO was measured from cª²wave peak to the FO trough while LP was measured from the FO trough to the LP maximum [18]. As shown in Figure 1C, ERG bª²wave disappeared in 60mg/kg NaIO3 group from 28 days treatment, which decreased gradually in 40mg/kg NaIO3 group from 3 days and vanished after 2 months. Thirty mg/kg NaIO3 could decrease almost all the ERG waves at 7, 14 and 28 days (Figure 1Dª²H). Twenty mg/kg NaIO3 decreased the bª²wave at 7 days but recovered afterward. Fifteen and 7.5mg/kg NaIO3 didn¬ðt suppress any ERG wave (Figure 1Dª²H).

¡¡¡¡Fundus Photos and Fluorescein Angiography Rats from different groups were chosen to take ocular fundus pictures and fluorescein angiography (FA).For FA, hyperfluoreª²scence in the whole retina were seen in 60mg/kg NaIO3 group as early as at 3 days, even no obvious changes were seen in fundus pictures at the time. Partial retinal hyperfluorescence could be seen at 3 days in both 40mg/kg and 30mg/kg NaIO3 groups; the former is much more severe. Hypofluorescence could be seen from peripheral retina with a longer time period. Yellow dots or scars could be seen as early as at 7 days in all three groups from peripheral to the central retina, which was related to the dose of NaIO3 (Figure 2). In 20mg/kg NaIO3 group, changes were not obvious till 28 days.

¡¡¡¡Histology RPE cells fell off and photo cells decrease could be seen at 60mg/kg NaIO3 from 3 days, and 40 mg/kg NaIO3 from 7 days. The melanin disappearance in RPE cells could be seen at 30mg/kg NaIO3 from 7 days. No obvious changes were seen in 20mg/kg NaIO3 groups (Figure 3).

¡¡¡¡Autofluorescence of Flatmount Autofluorescence of RPE cells could be seen in flatmount (Figure 4) induced by laser with measurement of confocal microscope. After treatment with 30 mg/kg NaIO3 or higher, small holes increased from 3 days, which indicated necrosis of RPE cells. In 20mg/kg NaIO3 group, small holes in flatmounts were found from 7 days, which were less as compared with larger dose groups.

¡¡¡¡Cell Proliferation The cell rate decreased after 48 hours treatment with 5, 30 and 100mg/L NaIO3 (Figure 5). Lower concentrations of NaIO3 had no effect.

¡¡¡¡Effects of Hydralazine We measured ERG cª²wave of rats after administration of 35mg/kg NaIO3 for 4 weeks. It was found that ERG cª²wave fell markedly to 31% of normal group in NaIO3 group (P<0.01). In 10g/L hydralazine+ NaIO3 group, ERG cª²wave fell down to 50% of normal group (P<0.05) which was a significant reversal to 61% of suppression of NaIO3 group (P<0.01).

¡¡¡¡Figure 1 ERG waves after NaIO3 injection to BN rats. aP<0.05, bP<0.01, vs Control(ÂÔ)

¡¡¡¡DISCUSSION

¡¡¡¡It was found in this study that the retina of BN rat was easily damaged by high doses of NaIO3, from neural retina layers to photoreceptor and RPE cell layers, which was not reversible (Figure 1C). From morphological data we could also note that 60mg/kg NaIO3 induced every severe retina toxicity. In lower doses, such as 20mg/kg NaIO3 or lower, not so much histology changes were found. Even though the functional decrease was observed at the beginning, which could be recovered later. So neither too high nor too low doses of NaIO3 could be suitable for the treatment of dryª²AMD animal model. Accordingly, 30mg/kg to 40mg/kg NaIO3 would be optimal to be used in this animal model. As far as action mechanism is concerned, larger doses of NaIO3 could destroy all layers of retina whereas lower doses only influence photoreceptors and RPE cells (Figure 3C). Only 30mg/L NaIO3 and higher concentration could suppress RPE cells(Figure 5) , which might indicate that melanin is important for the toxicity of NaIO3 in RPE cells. Since not much melanin exists in the cell cytoplasm, low dose of NaIO3 has no effect on APREª²19 cells growth.

¡¡¡¡Since hydralazine can cause choroidal vasodilatation and increase choroidal blood flow in the eyes, retina can receive more blood and oxygen. We found that for rats treated with 10g/L hydralazine after NaIO3 injection, the ERG cª²wave didn¬ðt fall as markedly as in NaIO3 group. This action of hydralazine might postpone the development of nonª²exudative ageª²related macular degeneration and could be used to treat nonª²exudative ageª²related macular degeneration in the future.

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