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Effects of hydralazine on NaIO3 induced rat retinal pigment epithelium degeneration

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作者:Wei Jiang    作者单位:Institute of Ocular Pharmacology, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843, USA

【摘要】    AIM: To study the effects of 10g/L hydralazine eye drops on 35mg/kg NaIO3 induced degeneration in rat eyes.

  METHODS: Various doses of NaIO3 and/or saline alone were injected into Brown Norway rats from hypoglossal vein. After 3, 7, 14 or 28 days of injection, ERG a, b, cwave, fast oscillation (FO) and light peak (LP) were measured along with retinal colored pictures and fluorescein angiography taken. Some rats were chosen to study the histology of retinas by light microscopy and autofluorescence of retina flatmounts. Different concentrations of NaIO3 were given to RPE19 cells, and cell proliferation rate was measured. For hydralazine study, 35mg/kg NaIO3 was injected into Brown Norway rat from hypoglossal vein. NaIO3 group was treated with saline alone after NaIO3 injection, 10g/L hydralazine+ NaIO3 group was treated with 10g/L hydralazine eyedrops after NaIO3 injection whereas normal group was treated with saline alone without NaIO3 injection. All eyedrops were instilled locally 3 times a day for 4 weeks and ERG cwave was measured at the end of 2 and 4 weeks.

  RESULTS: After NaIO3 administration, the amplitude of all ERG waves fell markedly in large dose groups at 30, 40 or 60mg/kg NaIO3. Not many changes were observed in groups treated with <30mg/kg NaIO3. Some retinal necrosis appeared from 3 days postinjection (PI) in 30mg/kg NaIO3 group, which became more serious in larger dose groups or longer treatment time, but no apparent change was found in smaller dose groups. Similarly, on the retina flatmount, RPE monolayer showed necrosis from 3 days PI in the 30mg/kg NaIO3 and larger dose groups. On histological examination, no significant change was seen in 30mg/kg NaIO3 and lower concentration groups. In cell culture experiment, changes were found in RPE19 cells proliferation rate with a concentration of NaIO3 at 30mg/L or higher. In hydralazine experiments, 4 weeks after injection of NaIO3, ERG cwave fell markedly in NaIO3 group to 31% of control group (P<0.01). The ERG cwave of hydralazine +NaIO3 group fell only to 50% of control group (P<0.05). This was a 61% reversal of the cwave of NaIO3 treated group.

  CONCLUSION: RPE degeneration induced by NaIO3 was both dose and time dependent. Around 30 to 40 mg/kg NaIO3 would be the optimal to be used as a nonexudative agerelated macular degeneration rat model. Hydralazine may postpone the development of nonexudative agerelated macular degeneration.

 

【关键词】  retinal pigment epithelium sodium iodate agerelated macular degeneration hydralazine

  INTRODUCTION

  Retinal pigment epithelium (RPE) monolayer plays a very important role in retinal function and many pathologic processes in eye diseases, especially the agerelated macular degeneration (AMD). In non exudative or ‘dry’ agerelated macular degeneration, dysfunction of RPE is the first step followed by lipofuscin accumulation, presence of drusen, RPE atrophy and loss of photoreceptors. For searching a good model of dryAMD, many people focused on sodium iodate, which could lead to RPE degeneration and atrophy selectively. Previous studies showed that after systemic injection of NaIO3, ocular fundus showed following changes [1,2]. First, degeneration of RPE cells which could be seen from the histology and the suppression of ERG cwave. It was then followed by the reduction of ERG a and b waves. The last part affected by NaIO3 was the inner retina.
  However, the results were inconsistent depending on doses, time period and species used. When ICR albino mice were injected with 40mg/kg of sodium iodate intravenously, a and bwave amplitudes decreased first but could recover to the normal levels in 4 hours after injection [3]. If Balb/c mice were injected with 40mg/kg of NaIO3 solution through the caudal vein, the suppressed bwave amplitude began to recover at 14 days and totally recovered at 6 weeks after injection [4]. When New Zealand albino rabbits were injected with 0.5mg/kg NaIO3, regeneration of RPE was noted at 6 to 7 days after NaIO3 administration [5].

  There are various explanations given to the different sensitivity of RPE cells to NaIO3 actions. One theory indicated that NaIO3 might act on melanin, a large component in RPE cells, which could be released from melanosome to convert glycine into glyoxylate[6]. Another study suggested that NaIO3 could denature retinal proteins manifested by changes of SH level in retina [7]. Another suggestion indicated that retinotoxic effect of NaIO3 was through inhibition of sulphydryl enzyme activity [8]. A group of articles considered the structural changes induced by sodium iodate, either via breakdown of retinal pigment epithelium diffusion barrier [9,10], or reduction of adhesion between RPE and photoreceptor cells [11,12]. As a strong oxidizing agent and selectively affecting RPE cells, NaIO3 was used as a model to evaluate drug actions on AMD or similar retinopathy diseases [1315].

  ERG measurement is widely used in various animal experiments. It is well known that, the ERG comprised two major components, the a and bwaves. The former reflects the function of rod photoreceptor outer segments [16] and the latter represents the activity of retinal bipolar cells [17]. Those two waves can be recorded by accoupled amplification. After bwave, a series of slow potentials follow to which dccoupled amplification is required [18]. Cwave is the second positive potential related to the transepithelial potential of the RPE. Fast oscillation (FO) has a negative trough that follows cwave which relates to the basal membrane of the RPE. Following FO is a positive trough named light peak (LP) which also relates to the basal membrane of RPE.

  It was tried to determine the morphological and functional changes in Brown Norway rats induced by various doses of NaIO3. During the study of various ERG waves, an optimal dose of NaIO3 was established in Brown Norway rats as a model to study the antiAMD drugs. Hydralazine is a vasodilator clinically used to treat hypertension. Furthermore, it has previously been shown in microdialysis experiments to cause vasodilatation[19,20]. Local instillation of hydralazine eye drops has been found to alter intraocular pressure in animal eyes. We used hydralazine to treat rats after NaIO3 injection and tried to observe effects of hydralazine eyedrops on reversal of NaIO3 induced RPE degeneration.

  MATERIALS AND METHODS

  Materials  8weekold male BrownNorway (BN) rats were purchased from LARR (Texas A&M University, USA). All rats were housed in a standard animal room for a 12∶12 hour cyclic lighting schedule. Animals were fed with normal food and water. All of the procedures conformed to the ARVO Resolution on the use of animals in ophthalmic and vision research. NaIO3 (SigmaAldrich) was dissolved by saline at a mass concentration of 30g/L. Single injection of different doses of NaIO3 (0, 7.5, 15, 20, 30, 40, 60mg/kg) was made through sublingual vein. Functional and histological changes examined at post injection (PI) 3 to 28 days or 2 months selectively. 10g/L hydralazine solution was prepared by Pam Louis Assoc. (San Antonio, TX).

  Animal Procedure  After single injection of NaIO3, rats in different groups were measured with ERG, fundus pictures and fluorescein angiography at different time periods from 3 days to 2 months PI selectively. After functional examination, some rats from different groups were sacrificed and the eyes were removed and fixed in 25g/L glutaraldehyde for 2 hours and then in 20g/L formaldehyde overnight. One eye of each animal was used for histology and immunohistology studies, the other eye was prepared for autofluorescence measurement on flatmounts. For hydralazine studies, normal group was instilled with saline alone without NaIO3 injection. NaIO3 group was instilled with saline alone after 35mg/kg NaIO3 injection, whereas 10g/L hydralazine+ NaIO3 group was instilled with 10g/L hydralazine eye drops after 35mg/kg NaIO3 injection. All eyedrops were instilled 3 times a day for 4 weeks. At the end, all rats in different groups were measured with ERG cwave.

  ERG Recordings  BN rats were dark adapted overnight, and then anesthetized with ketamine 35mg/kg plus xylazine 5mg/kg intramuscularly. Half of the initial dose was given to each 1 hour thereafter. Pupils of all rats were dilated with one drop of 10g/L atropine, 10g/L tropicamide and 25g/L phenylephrine. Before recording, one drop of opticaine was used for surface anesthetization. All animals were kept warm during ERG measurement. Each rat was measured by dcERG recording firstly then by acERG recording.

  ACERG Recording  When ERG was recorded, Ag/AgCl electrode was placed gently in contact with the cornea as a reference electrode. A drop of 9g/L NaCl was used between them to establish stable signals. One stainless steel long electrode was inserted beneath the foreheads skin between the two eyes and another stainless steel short electrode was inserted  to the leg as a ground electrode. A photostimulator (Grass PS22 Flash) was used to produce flashes of light five inches from the eye. EPIC2000 was purchased from LKC Technologies, Inc (Gaithersburg, MD). A single scotopic white flash (20ms duration) was used to elicit ERG a and bwaves. The intensity of the stimuli was 628 cds/m2 and bandpass filtered from 0.3 to 500Hz.

  DCERG 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s corneal surface completely. Another similar electrode placed on the surface of the other eye served as a reference lead. Responses were amplified (dc100Hz; gain=1000×; DP301, 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 fiberlite high intensity illuminator (DolanJenner 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) LS110 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 (TRC50EX; 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  Paraffinembedded tissues were sectioned at 3μm 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).

  ARPE19 Cells Culture  ARPE19 cell line was purchased from ATCC Company (American Type Culture ollection, Manassas, VA). It was incubated in 37℃, 50mL/L CO2 condition. Growth medium was composed of 1∶1 mixture of Dulbeccos modified Eagles medium (DMEM) and Hams F12 medium containing 1.2g/L sodium bicarbonate, 2.5mmol/L Lglutamine, 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 trypsin0.2g/L EDTA (SigmaAldrich, St. Louis, MO).

  Cell Proliferation Assay  ARPE19 cells were grown in 96well 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s phosphatebuffered saline one time and further incubated with 100g/L 3(4,5dimethythiazol2yl)2,5diphenyl tetrazolium bromide (MTT) 100μL for exactly 4 hours. Media was then removed by aspiration, 100μL 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. Onetail ttest was used for in vivo  experiment and twotail ttest was used for in vitro part.

  RESULTS

  ERG Recordings  After ACERG recording, we measured awave from baseline to the first negative trough; maximum bwave amplitude (Vbmax) from the first negative trough (awave) to the first positive peak of the b wave (Figure 1A). For the DCERG recording (Figure 1B), the second positive peak followed bwave was cwave. The amplitude of which was measured from the trough after bwave (which was after potential, AP) to the peak of cwave. The amplitude of FO was measured from cwave peak to the FO trough while LP was measured from the FO trough to the LP maximum [18]. As shown in Figure 1C, ERG bwave 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 1DH). Twenty mg/kg NaIO3 decreased the bwave at 7 days but recovered afterward. Fifteen and 7.5mg/kg NaIO3 didnt suppress any ERG wave (Figure 1DH).

  Fundus Photos and Fluorescein Angiography  Rats from different groups were chosen to take ocular fundus pictures and fluorescein angiography (FA).For FA, hyperfluorescence 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 cwave of rats after administration of 35mg/kg NaIO3 for 4 weeks. It was found that ERG cwave fell markedly to 31% of normal group in NaIO3 group (P<0.01). In 10g/L hydralazine+ NaIO3 group, ERG cwave 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(略)

  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 dryAMD 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 APRE19 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 cwave didnt fall as markedly as in NaIO3 group. This action of hydralazine might postpone the development of nonexudative agerelated macular degeneration and could be used to treat nonexudative agerelated macular degeneration in the future.

  Figure 2 Ocular fundus and fluorescein angiogram on NaIO3 treated BN rats (略)

  A: retinograph of 30mg/kg NaIO3 treated BN rat at day 28; B: fluorescein angiogram of 30mg/kg NaIO3 treated BN rat at day 28; C: retinograph of 40mg/kg NaIO3 treated BN rat at day 7; D: fluorescein angiogram of 40mg/kg NaIO3 treated BN rat at day 7; E: retinograph of 40mg/kg NalO3 treated BN rat at day 8

  Figure 3  Histology of retina on NaIO3 treated BN rat eyes (略)

  A: 20mg/kg NaIO3 at 28 days;B: 30mg/kg NaIO3 at 28 days;C: 40mg/kg NaIO3 at 14 days

  Figure 4  Autofluorescence of RPE cells in flatmount induced by laser and measured with confocal microscope(略) 
  
  A:normal RPE cells; B: 30mg/kg NaIO3 at 7 days; C: 40mg/kg NaIO3 at 7 days

  Figure 5  ARPE19 cells proliferation rate after 48hour treatment with NaIO3(略) 

  aP<0.05 ,bP<0.01, vs Control

  Figure 6  Effects of hydralazine eyedrops on 35mg/kg NaIO3(略) 

  induced RPE degeneration in rat eyes  aP<0.05,bP<0.01, vs Normal, dP<0.01, vs NaIO3

  Acknowledgements  The authors are grateful to Prof N.S. Peachey, J. Wu, and M. Yu for invaluable assistance in dcERG equipment assembling and measurement. Supported in party by MacuClear, Inc.

【参考文献】
    1 Nilsson SEG, Knave B, Persson HE. Changes in ultrastructure and function of the sheep pigment epithelium and retina induced by sodium iodate. Acta Ophthalmol1977;55(6):10271043

  2 Grignolo A, Orzalesi N, Calabria GA. Studies on the fine structure and the rhodopsin cycle of the rabbit retina in experimental degeneration induced by sodium iodate. Exp Eye Res 1966;5:8697

  3 Hosoda L,AdachiUsami E,Mizota A,Hanawa T,Kimura T.Early effects of sodium iodate injection on ERG in mice. Acta Ophthalmol(Copenh) 1993;71(5):616622

  4 Mizota A, AdachiUsami E. Functional recovery of retina after sodium iodate injection in mice. Vision Res 1997;37(14):18591865

  5 Korte GE, Wanderman MC. Distribution of Na+ K+ATPase in regenerating retinal pigment epithelium in the rabbit. A study by electron microscopic cytochemistry. Exp Eye Res 1993;56:219229

  6 Baich A, Ziegler M. The effect of sodium iodate and melanin on the formation of glyoxylate. Pigment Cell Res1992;5:394395

  7 Sorsby A, Reading HW. Experimental degeneration of the retinaⅪ.The effect of sodium iodate on retinalSH levels. Vision Res 1964;4:511514

  8 Sorsby A, Reading HW. Experimental degeneration of the retinaⅦ.The protective action of thiol donors against the retinotoxic effect of sodium iodate. Vision Res1962;2:139148

  9 Sen HA,Berkowitz BA,Ando N,de Juan E Jr. In vivo imaging of breakdown of the inner and outer bloodretinal barriers. Invest Ophthalmol Vis Sci 1992;33(13):35073512

  10 Flage T, Ringvold A. The retinal pigment epithelium diffusion barrier in the rabbit eye after sodium iodate injection. A light and electron microscopic study using horseradish peroxidase as a tracer. Exp Eye Res 1982;34(6):933940

  11 Ashburn FS, Pilkerton AR, Rao NA, Marak GE. The effects of iodate and iodoacetate on the retinal adhesion. Invest Ophthalmol Vis Sci 1980;19(12):14271432

  12 Stern WH, Ernest JT, Steinberg RH, Miller SS. Interrelationships between the retinal pigment epithelium and the neurosensory retina. Aust J Ophthalmol 1980;8(4):281288

  13 Ohtaka K, Machida S, Ohzeki T, Tanaka M, Kurosaka D, Masuda T, Ishii T. Protective effect of hepatocyte growth factor against degeneration of the retinal pigment epithelium and photoreceptor in sodium iodateinjected rats. Curr Eye Res 2006;31(4):347355
  
  14 Obata R, Yanagi Y, Tamaki Y, Hozumi K, Mutoh M, Tanaka Y. Retinal degeneration is delayed by tissue factor pathway inhibitor2 in RCS rats and a sodiumiodateinduced model in rabbits. Eye 2005;19(4):464468
  
  15 AtmacaSonmez P, Li Y, Yamauchi Y, Schanie CL, Ildstad ST, Kaplan HJ, Enzmann V. Systemically transferred hematopoietic stem cells home to the subretinal space and express RPE65 in a mouse model of retinal pigment epithelium damage. Exp Eye Res 2006;83(5):12951302

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