¡¡¡¡DCª²ERG Recording Methods developed by Dr. Peachey were followed[18]. Briefly, a 1mm diameter glass capillary tube with filament (Sutter Instruments, Novato, CA) that was filled with Hanks balanced salt solution (Invitrogen, Carlsbad, CA) was used to contact with a Ag/AgCl wire electrode with a attached connector. The capillary tube was connected with rat¬ðs corneal surface completely. Another similar electrode placed on the surface of the other eye served as a reference lead. Responses were amplified (dcª²100Hz; gain=1000¡Á; DPª²301, Warner Instruments, Hamden, CT) and digitized at 10Hz or 1000Hz. Data were analyzed by iWORX LabScribe Data Recording Software (iWorx0CB Sciences, Dover, NH). Light stimuli was derived from an optical channel using a fiberª²lite high intensity illuminator (Dolanª²Jenner Industries, MA), with neutral density filters (Oriel, Stratford, CT) placed in the light path to adjust stimulus luminance. The stimulus luminance used in this experiment was 3.22logcd/m2 and stimulated for 4 minutes. Luminance calibration was made by a Minolta (Ramsey, NJ) LSª²110 photometer focused on the output side of the fiber optic bundle where the rat eye was located.

¡¡¡¡Fundus Pictures and Fluorescein Angiography Digital fundus camera (TRCª²50EX; TOPCON, Tokyo, Japan) and Imagenet 2000 digital imaging systems (Topcon Medical Systems, Inc., Paramus, NJ) were used to capture retinal colored pictures and fluorescein angiography. When using fluorescein angiography, 10mg of fluorescein sodium was injected through the hypoglossal vein of rats. Anesthesia and pupil dilation were done as mentioned above.

¡¡¡¡Histology Paraffinª²embedded tissues were sectioned at 3¦Ìm thickness. Eyes were cut from cornea to the optic nerve head along the vertical meridian, then stained with hematoxylin and eosin. Axioskop microscope (Zeiss, Thornwood, NY) was used to capture the images.

¡¡¡¡Autofluorescence of Flatmount For preparation of flat mounts, one eye of each animal was enucleated. After fixation, anterior part of the eye, as well as cornea, lens and sensory retina were gently removed and the remaining eye cup was washed in PBS. Four cuts were made from edge to center which helps to flatten the eye cup onto a glass slide. The autofluorescence of RPE in the flatmount was studied and captured on a confocal microscope (Zeiss LSM510; Zeiss, Thornwood, NY) using an Argon laser (wavelength 488nm).

¡¡¡¡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)

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