【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, cwave, 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 concentrations of NaIO3 were given to RPE19 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 cwave 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 postinjection (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 RPE19 cells proliferation rate with a concentration of NaIO3 at 30mg/L or higher. In hydralazine experiments, 4 weeks after injection of NaIO3, ERG cwave fell markedly in NaIO3 group to 31% of control group (P<0.01). The ERG cwave of hydralazine +NaIO3 group fell only to 50% of control group (P<0.05). This was a 61% reversal of the cwave 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 nonexudative agerelated macular degeneration rat model. Hydralazine may postpone the development of nonexudative agerelated macular degeneration.
【关键词】 retinal pigment epithelium sodium iodate agerelated 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 agerelated macular degeneration (AMD). In non exudative or ‘dry’ agerelated 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 dryAMD, 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 cwave. 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 bwave 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 bwave 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 [1315].
ERG measurement is widely used in various animal experiments. It is well known that, the ERG comprised two major components, the a and bwaves. 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 accoupled amplification. After bwave, a series of slow potentials follow to which dccoupled amplification is required [18]. Cwave is the second positive potential related to the transepithelial potential of the RPE. Fast oscillation (FO) has a negative trough that follows cwave 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 antiAMD 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 8weekold male BrownNorway (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 (SigmaAldrich) 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 cwave.
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 dcERG recording firstly then by acERG recording.
ACERG 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 foreheads 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. EPIC2000 was purchased from LKC Technologies, Inc (Gaithersburg, MD). A single scotopic white flash (20ms duration) was used to elicit ERG a and bwaves. The intensity of the stimuli was 628 cds/m2 and bandpass filtered from 0.3 to 500Hz.
DCERG 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 rats corneal surface completely. Another similar electrode placed on the surface of the other eye served as a reference lead. Responses were amplified (dc100Hz; gain=1000×; DP301, 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 fiberlite high intensity illuminator (DolanJenner 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) LS110 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 (TRC50EX; 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 Paraffinembedded 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).
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