Neuroprotective Effect of Citicoline on Retinal Cell Damage Induced by Kainic Acid in Rats Yong Seop Han, MD, In Young Chung, MD, Jong Moon Park, MD, PhD, Ji Myeong Yu, MD, PhD Department of Ophthalmology, College of Medicine, Gyeongsang National University, Jinju, Korea Purpose: To examine whether citicoline has a neuroprotective effect on kainic acid (KA)-induced retinal damage. Methods: KA (6 nmol) was injected into the vitreous of rat eyes. Citicoline (500mg/kg, i.p.) was administered to the rats once before and twice a day after KA-injection for 3- and 7-day intervals. The neuroprotective effects of citicoline were estimated by measuring the thickness of the various retinal layers using hematoxylin-eosin (H&E) staining. In addition, immunohistochemistry was conducted to elucidate the expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS). Results: Morphometric analysis of retinal damage in KA-injected eyes showed significant cell loss in the inner nuclear layer (INL) and inner plexiform layer (IPL) of the retinas at 3 and 7 days after KA injection, but not in the outer nuclear layer (ONL). At 3 days after citicoline treatment, no significant changes were detected in the retinal thickness and immunoreactivities of eNOS and nNOS. The immunoreactivities of eNOS and nNOS increased in the retina at 7 days after the KA injection. However, prolonged treatment for 7 days significantly attenuated the immunoreactivities and the reduction of thickness. Conclusions: The results indicate that citicoline has a neuroprotective effect on KA-induced neurotoxicity in the retina. Korean Journal of Ophthalmology 19(3):219-226, 2005 Key Words: Citicoline, Kainic acid, Neuroprotective effect, NOS Retinal ischemic injuries are known to be associated with increases oxygen consumption.13-15 The generation of nitric excitotoxicity of glutamate.1,2 It is generally believed that oxide (NO) and NOS following the process described above ischemia caused by elevated intraocular pressure (IOP), triggers the fragmentation of DNA, which in turn leads to which elicits mechanical damage to the retinal cells or retinal ganglion cell death through the elevation of p53.16 impedes blood flow, plays an important role in the mecha- NO mediates several biological actions, including relax- nism of how retinal ganglion cell death occurred.3-7 In ation of blood vessels and cytotoxicity, and acts as gaseous addition to retinal ischemia, excitotoxicity of glutamate, lack neurotransmitters in the central and peripheral nervous of neurotrophin and oxidative damage are also discussed as system.17,18 Also, it plays an important role in neuronal possible causes of retinal ganglion cell death.8,9 damage in the CNS following brain ischemia, and the In a hypoxic state, there is a large release of the excitatory administration of NOS inhibitors protected the retina from neurotransmitter, glutamate, into the extracellular space10-12 degenerative changes and provided protection of retinal and resultant glutamate excitotoxicity, which is promoted by ganglion cells from ischemic damage.19-23 N methyl D aspartate (NMDA) and kainate receptors. The Citicoline (Cytidine-5-diphosphocholine, exogenous CDP- excitotoxicity induced by kainic acid (KA), a glutamate choline), first synthesized by Eugene Kennedy in 1956, has analogue, involves a process in which AMPA/KA receptors proved effective in treating a wide range of cognitive are overstimulated, which in turn leads to Na＋ influx, cell disorders caused by CNS ischemia, brain injury and depolarization and eventually excessive influx of Ca2＋ as the Parkinson's disease.24,25 Endogenous CDP-choline is a key voltage-dependent Ca2＋ channel become permeable to Ca2＋. intermediary in the biosynthesis of phosphatidylcholine Excessive accumulation of Ca2＋ by neural cells activates (PtdCho). Increased biosynthesis of PtdCho prevents neuronal cellular proteases and nitric oxide synthase (NOS) and apoptosis and promotes neuroprotection. Citicoline is also known to preserve cardiolipin and sphingomyelin, stimulate glutathione biosynthesis, increase the activity of glutathione Received: May 12, 2005 Accepted: August 25, 2005 24,25 reductase and reduce lipid peroxidation. Reprint requests to Jong Moon Park, MD. Department of Ophthal- mology, College of Medicine, Gyeongsang National University, #92 This study aimed to evaluate the neuroprotective effects of Chillam-dong, Jinju, Gyeong-nam 660-280, Korea. Tel: 82-55-750- citicoline by analyzing the morphometric features of cells and 8170, Fax: 82-55-758-4158, E-mail: [email protected] the expression of NOS in the adult rat retina after KA- 219 Kor J Ophthalmol Vol.19, No.3, 2005 induced injuries. nitrogen and then stored at -70℃ until sectioning. The tissues were kept at -20℃ for an hour for sectioning Materials and Methods into 12 µm-thick continuous frozen sections with a cryostat (Leica, Meyer Instruments, Inc. Houston, TX). The sections 1. Sample animals preparation were mounted on gelatin-coated slides, dried for 30 minutes at 42℃ and stored at -70℃ until use. Sprague-Dawley (SD) white male rats (Samtaco, Osan, Korea) weighing 200-250 g were maintained in cages under 5. Morphometric features a 12-hour light and 12-hour dark cycle for a week. A total of 60 rats were divided into three groups of 20 rats each: The prepared frozen sections were kept at room rats treated with KA only, rats treated with citicoline one temperature for about an hour, dehydrated to increase the hour prior to and twice a day after the administration of KA, stickiness between the section and the slide, washed in two and normal rats (the control group). Each group were changes of distilled water for 5 minutes each and dehydrated subdivided into 3- and 7-day subgroups according to time again by water deprivation. After dehydrating, the sections duration after injection. were stained in 0.5% hematoxylin for 5 minutes, washed in two to three changes of distilled water, soaked in hydro- 2. A rat model for the study of Kainic acid-induced chloric acid for a second time and then washed in several retinal damage changes of distilled water to enhance the color after hematoxylin staining. The hematoxylin-stained sections were Each rat was intraabdominally anesthetized with a double-stained with 0.6% eosin for 2 minutes, dehydrated combination of a 30 mg/kg dose of ketamine (Yohan Keta- through a graded alcohol series (1 minute each in 70%, 80%, mine, Yohan Corporation) and a 2.5 mg/kg dose of xylazine 90%, 95%, and 100%), cleared in xylene series and mounted (Yuhan Rompun, Yuhan Corporation). The anesthetized rat permanently. Stained tissue slides were examined using a was immobilized in a stereotaxic frame and a drop of microscope and pictures were taken. To examine the morpho- ofloxacin was administered into the eye to avoid infection. metric changes of the retina after administrating KA and KA (Sigma, USA) was dissolved in 2 mM, 3 ul (6 nM) citicoline, the layer-structure of the retina thickness and of which was injected into the vitreous of the rat eyes using retinal cells in treated and control rats were assessed and 26 a 30 gauge microsyringe. An ophthalmoscopic examination compared at the location of 800-1,000 µm from the optic was performed to ensure that KA was accurately injected into nerve in five slides per rat. the vitreous of the rat eyes, after which ofloxacin was once again applied. 6. NOS immunohistochemistry 3. Citicoline administration The prepared frozen sections were kept at room temperature for about an hour and dehydrated by water After KA injection, rats were housed in separate cages deprivation. After dehydrating, the sections were washed in according to the subclassification of the 3- and 7-day groups. 0.02M phosphate buffer saline (PBS), 0.5% Triton Citicoline-treated rats received 500 mg/kg citicoline intra- X-100/0.02 M PBS and 0.02 M PBS for 15 minutes each, abdominally an hour prior to and twice a day at 12-hour treated in 0.3% H2O2 for 30 minutes to inactivate endogenous intervals after KA injection. This procedure was routinely peroxidase, and washed again in the same way as described performed without anesthesia. above. After washing, normal serum was applied to the slides, which were then incubated for 30 minutes. After 4. Tissue isolation normal serum was cleaned up, each primary antibody was applied to the slides, which were then incubated for 16 to On days 3 and 7 after KA injection, 10 rats in each sub- 18 hours at 4℃. For the primary antibodies against neuronal group were sacrificed and the eyes were rapidly enucleated. NOS (nNOS) and endothelial NOS (eNOS), mouse anti- The cornea was removed promptly from the enucleated nNOS monoclonal and mouse anti-eNOS polyclonal anti- eyeballs and then placed into 4% neutralized phosphate bodies were used. Anti-nNOS and anti-eNOS primary anti- buffered paraformaldehyde (NBP). After an hour, the lens bodies were applied at a dilution of 1:100. Unbound primary was removed and the eyeballs were fixed in the same NBP antibody was washed off with 0.02 M PBS, 0.5％ Triton for 5 hours, after which the eyeballs were washed in 0.1 M X-100/0.02 M PBS and 0.02 M PBS, for 15 minutes each. PB (phosphate buffer), and soaked for 24 hours in 30% PB The sections were subsequently reacted with secondary sucrose to prevent irreversible damage to the tissues. After antibody (biotinylated anti-mouse IgG) for 90 minutes at soaking, sucrose solution was carefully washed off the tissue room temperature. After unbound secondary antibody was surface, and the tissues were embedded in OCT compound washed off in the same way as described above, the slides (Tissue-TekⓇ), frozen in isopentane cooled with liquid were reacted for 90 minutes at room temperature using an 220 YS Han, et al. NEUROPROTECTIVE EFFECT OF CITICOLINE Avidin Biotin Complex kit (Vectastain, Vector Labs.