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Neuroprotective effect of citicoline against KA-induced neurotoxicity in the rat retina
Experimental Eye Research 81 (2005) 350–358 www.elsevier.com/locate/yexer
Neuroprotective effect of citicoline against KA-induced neurotoxicity in the rat retina Chang Hwan Parka, Yoon Sook Kima, Hae Sook Noha, Eun Woo Cheonb, Young Ae Yangd, Ji Myong Yooc, Wan Sung Choia, Gyeong Jae Choa,* a
Department of Anatomy and Neurobiology, Institute of Health Science, College of Medicine, Gyeongsang National University, 92 Chilam-dong, Jinju, Kyungnam 660-751, South Korea b Division of Food Science, Jinju International University, Jinju, Kyungnam 660-845, South Korea c Department of Ophthalmology, Institute of Health Science, College of Medicine, Gyeongsang National University, 92 Chilam-dong, Jinju, Kyungnam 660-751, South Korea d Department of Occupational Therapy, College of Biomedical science and Enginnering, Inje University, GimHae, 621-749, South Korea Received 22 October 2004; accepted in revised form 15 February 2005 Available online 25 March 2005
Abstract We examined whether citicoline has neuroprotective effect on kainic acid (KA)-induced retinal damage. KA (6 nmol) was injected into the vitreous of rat eyes. Rats were injected intraperitoneally with citicoline (500 mg kgK1, i.p.) twice (09:00 and 21:00) daily for 1, 3 and 7 days after KA-injection. The neuroprotective effects of citicoline were estimated by measuring the thickness of the various retinal layers. In addition, immunohistochemistry was conducted to elucidate the expression of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH). Morphometric analysis of retinal damage in KA-injected eyes showed a significant cell loss in the inner nuclear layer (INL) and inner plexiform layer (IPL) of the retinas at the 1, 3 and 7 days after KA injection, but not in the outer nuclear layers (ONL). At 1 and 3 days after citicoline treatment, no significant changes were detected in the retinal thickness and immunoreactivities of ChAT and TH. The immunoreactivities of ChAT and TH had almost disappeared in the retina after 7 days of KA injection. However, prolonged citicoline treatment for 7 days significantly attenuated the reduction of retinal thickness and immunoreactivities of ChAT and TH. The present study suggests that treatment with citicoline has neuroprotective effect on the retinal damage due to KA-induced neurotoxicity. q 2005 Elsevier Ltd. All rights reserved. Keywords: choline acetyltransferase; tyrosine hydroxylase; kainic acid; citicoline; retina; rat
1. Introduction Glutamate is the main excitatory neurotransmitter in the CNS. In the retina, L-glutamate is highly concentrated in the photoreceptor, the bipolar and ganglion cell layer (Hara and Sukamoto, 1993; Berger et al., 1997; Wu and Maple, 1998). Intravitreal injection of kainic acid (KA), a structural analogue of L-glutamate, induces rapid and selective lesions in the inner retina of rats with the photoreceptor cell-sparing (Goto et al., 1981; McGeer, 1982); * Corresponding author. Dr Gyeong Jae Cho, Department of Anatomy and Neurobiology, College of Medicine, Gyeongsang National University, 92 Chilam-dong, Jinju, Kyungnam 660-751, South Korea. E-mail address: [email protected] (G.J. Cho).
0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.02.007
it is well known that glutamate receptor-related neurotoxicity can be induced by NMDA (N-methyl-D-aspartic acid) and KA (Ehrlich and Morgan, 1980; Morgan and Ingham, 1981; Siliprandi et al., 1992). Neuronal damage, followed by cell loss, also has been observed in the retinas exposed to relatively high concentrations of glutamate and glutamate analogues, such as activating NMDA receptors or KA/AMPA (kainic acid/a-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid) receptors (Lucas and Newhouse, 1957; Olney, 1969; Yazulla and Kleinschmidt, 1980; Hampton et al., 1981; Abrams et al., 1989; Osborne and Quack, 1992; Perez and Davanger, 1994; Secades and Frontera, 1995). An overstimulation of these receptors leads to increased intracellular Ca2C levels and constitutes one of the steps in the development of the excitotoxic retinal cell damage which is observed following ischemia (Bresnick, 1989).
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Citicoline, a naturally occurring endogenous nucleoside, also known as CDP-choline (cytidine-5-diphosphocholine), is one of the rate-limiting factor in the formation of phosphatidycholine (PtdCho), an essential phospholipid for the maintenance of intracellular and extracellular membranes in the brain (Kennedy and Weiss, 1956; Trovarelli et al., 1981). Citicoline crosses the blood–brain barrier as cytidine and choline, in rodents, but not in humans, where its circulating breakdown products are uridine and choline (Wurtman et al., 2000), which reach the brain and synthesize again the citicoline in the cytoplasm (Secades and Frontera, 1995). Citicoline and its hydrolytic products (cytidine and choline) play important roles in the generation of phospholipids, which are involved in membrane formation and repair; cytidine and choline also contribute to critical metabolic function such as the formation of nucleic acids, proteins and acetylcholine (Weiss, 1995). For these reasons, citicoline has been assayed as a therapeutic agent in a variety of central nervous system (CNS) injury models and neurodegenerative diseases. Further, a few clinical studies showed beneficial effects of citicoline on the function of the visual pathway in patients with retinal damage (Parisi et al., 1999). In the rat retina, amacrine cells mediate lateral interaction in the inner retina. They are involved in modifying receptive field properties of ganglion cells and temporally modulating signal transfer from the bipolar cells to the ganglion cells (MacNeil and Masland, 1998); the dopaminergic amacrine cells are associated with retinal adaptation, whereas the cholinergic amacrine cells are important for directional selectivity of a population of ganglion cells and contain ionotropic excitatory amino acid receptors. We investigated the retinal dopaminergic and cholinergic systems using tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in the rat retinas of KA-induced damage and showed neuroprotective action of citicoline in those cells by immunohistochemistry and morphometric analysis.
2. Materials and methods All animal experiments were conducted in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. 2.1. Animal models Male Sprague–Dawley rats (200–250 g) were maintained under controlled conditions of temperature (25 8C) and photocycle (12 hr on/12 hr off), and allowed free access to food and water. The animal number used in this experiment was 35 and divided into the three groups as control group (nZ5) and the two experimental groups (nZ15, each); KA injection group (1 day, 3 and 7 days, nZ5, each time point)
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and KA injection plus treatment with citicoline group (1 day, 3 and 7 days, nZ5, each time point). Rats were injected intraperitoneally with citicoline (500 mg kgK1, i.p.) twice (09:00 and 21:00) daily for 1, 3 and 7 days after KA-injection. For KA injection each animal was anesthetized with an intraperitoneal injection of a mixture of ketamine (30 mg kgK1; Yuhan Corporation, Seoul, Korea) and xylazine (2.5 mg kgK1; Bayer, Seoul, Korea) and placed in a stereotaxic frame. KA (sigma, USA) was dissolved in sterile normal saline. After topical application of 0.5% proparacaine hydrochloride (0.5% Alcaine; Alcon, USA) to the right eye, a single injection of 3 ml of 2 mM KA (total amount, 6 nmol) was done into the vitreous space with a microsyringe (Hamilton, Reno, NV) when the needle tip reached the midvitreous. These procedures were performed under an operating microscope. Gentamicin ophthalmic ointment was applied topically to the eye after cannulation of the vitreous space. 2.2. Tissue preparation The animals were sacrificed at 1, 3 or 7 days after KA injection and KA injection plus treatment with citicoline. The eyes were enucleated for morphologic and immunohistochemical (IHC) analysis. Immediately after enucleation, the eyes were cut open by a circular incision and the posterior eyecup was fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) for 6 hr at 4 8C, then rinsed in 0.1 M PB for 30 min at 4 8C. Tissues were rinsed in 30% sucrose in 0.1 M PB for 12 hr, frozen in embedding medium (O.C.T. Compound, Miles Inc., Elkhart, IN) in liquid nitrogen, and cryosectioned at a thickness of about 12 mm in a sagittal direction through the optic nerve using a cryostat (Leica 8400E; Leica, Tokyo, Japan). The sections were thaw-mounted on slides coated with 1% gelatin solution and stored at K70 8C until morphologic and immunohistochemical use. Some sections were stained with hematoxylin and eosin. Mounted sections were microscopically observed and photographed at the same magnification, and the images at a distance of approximately 0.8–1 mm from the optic nerve head of both control and experimental groups were measured. 2.3. Immunohistochemistry Retinal cryosections from control, KA injection and KA injection plus treatment with citicoline groups were rinsed in 0.02 M phosphate-buffered saline (PBS), pH 7.4, 0.5% Triton X-100 in 0.02 M PBS and again in 0.02 M PBS for 10 min each at room temperature before incubation in the primary antibody. They were then incubated with 3% normal goat or rabbit serum (Vector Labs, Burlingame, CA, USA), 5% bovine serum albumin (Gibco Labs, Grand Island, NY, USA), and 0.3% Triton X-100 in 0.02 M PBS for 30 min at room temperature. The following primary antibodies were used: polyclonal goat anti-choline
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Fig. 1. Representative light microscopic photographs of the rat retina under different experimental conditions. (A) Normal retina. Five well-organized retinal layers are seen; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer, GCL, ganglion cell layer. Histological features of transverse sections in the retina after 1 day (B), 3 days (D) and 7 days (F) of KA injection. In (B), the thickness of the retina is reduced due to the reduction of the inner retina (INL and IPL) as compared with the normal retina. In (D and F), the thickness of the retina is reduced due to the disappearance of the IPL and the INL. Histological features of transverse sections in the retina after 1 day (C), 3 days (E) and 7 days (G) of KA injection plus treatment with citicoline. Compared with those shown in (D and F), the thickness of the INL and IPL (E and G) increased. Calibration bar: 50 mm.
acetyltransferase (anti-ChAT) (1:100; Chemicon International Inc., Temecula, CA, USA), polyclonal rabbit anti-tyrosine hydroxylase (anti-TH) (1:200; Pel-Freez Biologicals, Rogers, Ark., USA), each in 3% normal goat or rabbit serum, 5% bovine serum albumin, and 0.3% Triton X-100 in 0.02 M PBS incubation solution. All incubations were performed overnight at 4 8C in a humidity-tight box, without agitation. To control non-specific binding of the antiserum, some tissue sections were incubated in antibodyfree incubation solution. The sections were then washed with PBS and Triton X-100 as described above. Secondary antibodies (affinity-purified biotinylated goat anti-rabbit or
rabbit anti-goat IgG (HCL), Vectastain ABC kit, Vector Labs) were diluted (1:200) in a incubation solution. The sections were exposed to secondary antibodies for 1.5 hr at room temperature, washed in PBS and Triton X-100 and incubated with avidin–biotin complex (Vectastain ABC kit, Vector Labs, diluted (1:50) in 0.02 M PBS) for 1.5 hr at room temperature. They were washed again in PBS and Triton X-100, rinsed in 0.05 M Tris–HCl buffer (pH 7.5) for 10 min at room temperature, and pre-incubated in 0.05% 3, 3 0 -diaminobenzidine tetrahydrochloride (DAB) in TB for 10 min with gentle rocking. Hydrogen peroxide was added to the same DAB solution to make a final concentration of
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0.01% H2O2. After 1–2 min, determined by the degree of staining using a light microscope (Olympus), the reaction was terminated with several washes in 0.002 M PBS. 2.4. Statistical analysis The results are the mean of results from five adjacent areas within 1 mm of the optic nerve. All measurements were analyzed using an image analysis program (Soft Imaging system GmbH, Germany). For accurate analyses of the decreased TH- and ChAT-positive amacrine cells, we selected sectioned retinas with intact morphology to both ends of the ciliary bodies, centering on the optic nerve. THand ChAT-positive amacrine cells in the INL and/or IPL were then counted per retina, and normal and experimental groups were compared. In particular, the number of ChATand TH-like immunoreactive amacrine cells in the GCL and INL were counted on five adjacent retinal sections of individual animals. Values were given as meanGS.E. Statistical significance of difference between groups was performed using an unpaired Student’s t-test.
3. Results 3.1. Morphometric analysis for neuroprotective effect of citicoline Intravitreal injection of KA caused neuronal damage to retinal tissue. In the KA-treated retina, the entire retinal thickness decreased gradually compared with the control retinas. Specifically, the thickness of the retina after 7 days of KA injection was reduced to about 43% of the control retinas (Figs. 1 and 2). Fig. 2 shows the changes in the thickness of the major retinal layers in controls and KAtreated rats at various time points after KA injection. The effects on the individual retinal cell layers were different with respect to the time-course and extent of thickness reduction (Fig. 2). The thickness of the inner plexiform layer (IPL) started to decrease in the retina after 1 day of KA injection (Fig. 1B). In the retina after 3 days of KA injection, the thickness of the IPL and inner nuclear layers (INL) was gradually diminished (Fig. 1D). At 7 days, there was a significant decrease in overall retinal thickness, with a marked thinning of the IPL and the INL, whereas the thickness of outer retinal layers (ONL) was changed only slightly (Fig. 1F). The protective effects of citicoline on the KA-induced neuronal damage were examined by measuring the thickness of the ONL, INL and IPL. In the citicoline treated retina, after 3 and 7 days of KA injection, there was a significantly attenuated reduction in the thickness of the INL and IPL caused by KA treatment (Figs. 1 and 2). The retinal layers of the citicoline-treated retina showed relatively wellmaintained structures compared with untreated retinas.
Fig. 2. Morphometric analysis of KA-induced changes in the thickness of the outer nuclear layer (ONL, A), inner nuclear layer (INL, B) and inner plexiform layer (IPL, C) of the untreated and citicoline-treated rat retinas. Values were measured in retinal sections shown in Fig. 1. The degree of reduction in the thickness of the retina was quantified by the thickness (mm) of retinal layers such as the ONL, INL and IPL at a distance of 1.0–1.5 mm from optic disc. The thickness of the retinal layers gradually diminished in the inner retina (INL, *P!0.05 and IPL, **P!0.001) of citicoline treated group as compared with the normal retina after KA injection (B and C); the reduction was significantly attenuated when animals were treated with citicoline. The results are shown as meanGS.E. Statistical significances were calculated by Student’s t-test between control and experimental retinas (*P!0.05).
3.2. Effect of citicoline on ChAT immunoreactivity in the retina after KA injection The effect of citicoline on ChAT expression in the retina at different time points after KA injection is shown by
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Fig. 3. Photomicrographs of choline acetyltransferase (ChAT) immunoreactivity of controls (A); after 1 day (B), 3 days (D) and 7 days (F) of KA injection and after 1 day (C), 3 days (E) and 7 days (G) of KA injection plus treatment with citicoline. In the control retinas (A), ChAT immunoreactivity appeared as two clearly defined bands (arrow) in the inner plexiform layer with cell bodies (arrowheads) in the INL and GCL. ChAT immunoreactivity almost disappeared after 7 days of KA-injection (F). Prolonged citicoline treatment prevented the reduction in the ChAT immunoreactivity induced by KA injection (G, arrows). Calibration bar: 50 mm.
immunohistochemical analysis (Fig. 3). In the control retina, ChAT immunoreactive cells were associated with two types of certain amacrine cells located in both the INL and the GCL, and two clear laminae (arrow) in the IPL (Fig. 3A). ChAT immunoreactivity started to decrease after 1 day of KA injection (Fig. 3B). The immunoreactivity of ChAT in retinas subjected to KA-injection showed clear changes when compared with the control retinas. The normal immunoreactivity in the IPL was reduced in intensity and often appeared as a single band (Fig. 3D). The retina after 7 days of KA injection displayed an almost complete disappearance of ChAT immunoreactivity (Fig. 3F), but the ChAT immunoreactivity was significantly protected by a treatment with citicoline for 7 days (Fig. 3G).
The number of ChAT-positive cell in the inner nuclear layer (INL) and ganglion cell layer (GCL) were counted. The number of ChAT-positive cells gradually decreased compared to the control retinas as time passes after KA injection (Fig. 4; **P!0.001, respectively; nZ5). 3.3. Effect of citicoline on tyrosine hydroxylase immunoreactivity in the retina after KA injection Tyrosine hydroxylase (TH) immunoreactivities in control, citicoline-treated and non-treated rat retinas are shown in Fig. 5. The immunoreactive amacrine cells consisted of two types, a small and a large type and the cell somata were located in the outer part of the INL with branches oriented
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Fig. 4. Cell counts of ChAT-positive cells. The number of ChAT-positive cell in the inner nuclear layer (INL) and ganglion cell layer (GCL) were counted. The number of ChAT-positive cells gradually decreased compared to the control retinas as time passes after KA injection. The retina showed an almost disappearance of ChAT-positive cells after 7 days of KA injection. Prolonged citicoline treatment for 7 days significantly protected the reduction in the number of ChAT-positive cells. Statistical significances were calculated by Student’s t-test between control and experimental retinas (**P!0.001).
mainly towards the inner layers in the retina. The processes emerging from these cells ramified mainly in the outer part (sublamina 1, small arrow) of the IPL (Fig. 5A). The retinal TH immunoreactive cells were sparsely distributed throughout the retinas after 1 and 3 days of KA injection (Fig. 5B and D). The retina showed an almost complete disappearance of TH immunoreactivities after 7 days of KA injection (Fig. 5F). In the retinas after 1 and 3 days of citicolinetreatment, the immunoreactivities of TH were not changed significantly compared with KA-treated retinas (Fig. 5C and E). However, prolonged citicoline treatment for 7 days prevented the entire disappearance of TH immunoreactivities induced by KA injection (Fig. 5G). We counted all large or small TH-positive amacrine cells in the INL–IPL margin per retina and carried out a comparative analysis between the control and experimental groups. TH-amacrine cells gradually reduced in the KA injection groups compared to controls, showing a 28, 53 and 90% decrease at 1, 3 and 7 days after KA injection, respectively (Fig. 6; **P!0.001, respectively; nZ5).
4. Discussion The present study demonstrated that citicoline has a neuroprotective effect on experimental models of KAinduced retinal damage. We found that citicoline treatment twice daily for 7 days prevented the loss of retinal ChAT and TH immunoreactivities, as well as the thinning of the inner retinal layers resulting from KA-injection. Injection of KA to the rat eye caused a destruction of the retinal neurons, which was counteracted by continuous treatment with
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citicoline for 7 days. This study clearly shows the change of inner retinal thickness in the retina of KA-induced damage model. The retinal cell damage occurred in the inner retina up to 1 day after KA injection, whereas cells in the ONL were damaged later. Morphological evaluation of transverse sections of retina with KA injection demonstrated a dose-dependent loss of cells in the INL and a reduction in the thickness of the IPL (Morgan and Ingham, 1981); low doses (6–20 nmol) of kainic acid reduce the content of the amacrine and horizontal cell markers acetylcholine and gamma-amino-butyric acid (GABA); higher doses of kainic acid lead to disappearance of both the outer and inner plexiform layers. The KA-dose in this study was 6 nmol, comparable to low dose of the previous study and our results are in good accordance with the finding of this report where amacrine acetylcholine was reduced compared to control. Clinical reports indicate that citicoline may have a therapeutic effect in patients with glaucoma. Some researchers reported that citicoline ameliorated ganglion cells death in vitro (Oshitari et al., 2002). However, the precise effect of citicoline on damaged retinal ganglion cells (RGCs) remains to be explained. Previous studies have shown that the KA-induced retinal ganglion cell death appears to be mediated via NMDA receptors, suggesting that KA toxicity is mediated by release of glutamate, which in turn activates NMDA receptors. KA increases the proportion of retinal ganglion cells that die, but the toxicity (due to both KA and the endogenous toxin) is totally prevented by 2-amino-5-phosphonovalerate (APV), a specific NMDA receptor antagonist (Sucher et al., 1991). Although the exact mechanisms by which citicoline produces its neuroprotective effects are unknown, it has been shown to reduce neuronal degeneration by inhibiting the apoptotic pathway induced by glutamate (Mir et al., 2003). Citicoline may have the neuroprotective action in glutamate-mediated cell death. ChAT is a reliable marker for cholinergic amacrine cells, which are located on either side of the IPL, in the INL and GCL of the rat retina. The dendrites of the ChAT immunoreactive cells are located as two distinct strata in the IPL. The protective effect of citicoline on the rat retina was demonstrated by an analysis of ChAT immunoreactive cells. We found that by 7 days after KA injection there was an almost complete disappearance of ChAT-positive cells. It is known that ChAT positive amacrine cells in the retina are highly vulnerable to intraocular injection of neurotoxins (Gomez-Ramos et al., 1985). So it is tempting to speculate that the vulnerability of ChAT-positive cells in the KAtreated retina may be due to the presence of kainate/AMPA and NMDA receptors on those neurons (Brandsta¨tter et al., 1994; Osbone et al., 1995; Peng et al., 1995). It is likely that the disappearance of ChAT immunoreactivity might be due to an excessive influx of calcium from an overactivation of ionotropic glutamate receptors (Louzada-Junior et al., 1992). In the present study, our results demonstrated that
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Fig. 5. Photomicrographs of tyrosine hydroxylase (TH) immunoreactivity of controls (A); after 1 day (B), 3 days (D) and 7 days (F) of KA injection and after 1 day (C), 3 days (E) and 7 days (G) of KA injection plus treatment with citicoline. In the control retinas (A), the immunoreactivity amacrine cell consisted of two types, a small (open arrow) and a large type (solid arrow). The retina showed an almost complete disappearance of TH immunoreactivity after 7 days of KA injection (F). This loss was significantly protected by prolonged citicoline-treatment, showing apparent TH immunoreactivity (G, arrowhead). Calibration bar: 50 mm.
citicoline-treatment counteracted the reduction of the ChAT immunoreactivity caused by kainic acid injection, showing that citicoline can act as a neuroprotective agent against kainic acid-induced retinal damage. Dopaminergic cells contain tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and these cells react with antibody specific for TH. In the rat retina, the majority of dopaminergic cells are located in the innermost sublayer of the INL. It was previously reported that there are two types of TH immunoreactive cells in the adult rat retina (Wu and Cepko, 1993; Martin-Martinelli et al., 1994). Type I cells are large, located in the INL and react strongly with anti-TH. Their dendrites ramify at the junction between the INL and the IPL. Type II cells are small and only slightly reactive with anti-TH. They are
located more deeply within the INL (Martin-Martinelli et al., 1989; Martin-Martinelli et al., 1994; Yoles and Schwartz, 1998). The results are consistent with these reports. Intraocular injection of KA, a powerful glutamate receptor agonist, induced a significant decrement in the specific activities of tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and degeneration of cells in the inner nuclear layer of the retina (Schwarcz and Coyle, 1977). Citicoline was shown to stimulate tyrosine hydroxylase activity and dopamine release (Secades and Frontera, 1995). Thus, neuroprotection following citicoline treatment in KA-induced retinal damage may be the consequence of stimulating in cholinergic and dopaminergic system. In conclusion, the results demonstrate that the citicoline can spare cells from KA-induced retinal damage, suggesting
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Fig. 6. Cell counts of TH-positive cells. TH-positive amacrine cells in the INL were counted. The number of TH-positive cells gradually decreased compared to the control retinas as time passes after KA injection. The retina showed an almost disappearance of TH-positive cells after 7 days of KA injection. Prolonged citicoline treatment for 7 days significantly protected the reduction in the number of TH-positive cells. Statistical significances were calculated by Student’s t-test between control and experimental retinas (**P!0.001).
that citicoline is an efficient drug for the treatment of experimental neurodegenerative retina wherein cholinergic and dopaminergic neurons are related.
Acknowledgements This Research was supported by a grant (M103KV010020 03K2201 02010) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of Republic of Korea and partially supported by a grant of the BK 21 Project of Ministry of Education.
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Wurtman, R.J., Regan, M., Ulus, I., 2000. Effect of oral CDP-choline on plasma choline and uridine levels in humans. Biochem. Pharmacol. 60, 989–992. Yazulla, S., Kleinschmidt, J., 1980. The effects of intraocular injection of kainic acid on the synaptic organization of the goldfish retina. Brain Res. 182, 287–301. Yoles, E., Schwartz, M., 1998. Elevation of intraocular glutamate levels in rats with partial lesion of the optic nerve. Arch. Ophthalmol. 116, 906–910.
Neuroprotective effect of citicoline against KA-induced neurotoxicity in the rat retina Chang Hwan Parka, Yoon Sook Kima, Hae Sook Noha, Eun Woo Cheonb, Young Ae Yangd, Ji Myong Yooc, Wan Sung Choia, Gyeong Jae Choa,* a
Department of Anatomy and Neurobiology, Institute of Health Science, College of Medicine, Gyeongsang National University, 92 Chilam-dong, Jinju, Kyungnam 660-751, South Korea b Division of Food Science, Jinju International University, Jinju, Kyungnam 660-845, South Korea c Department of Ophthalmology, Institute of Health Science, College of Medicine, Gyeongsang National University, 92 Chilam-dong, Jinju, Kyungnam 660-751, South Korea d Department of Occupational Therapy, College of Biomedical science and Enginnering, Inje University, GimHae, 621-749, South Korea Received 22 October 2004; accepted in revised form 15 February 2005 Available online 25 March 2005
Abstract We examined whether citicoline has neuroprotective effect on kainic acid (KA)-induced retinal damage. KA (6 nmol) was injected into the vitreous of rat eyes. Rats were injected intraperitoneally with citicoline (500 mg kgK1, i.p.) twice (09:00 and 21:00) daily for 1, 3 and 7 days after KA-injection. The neuroprotective effects of citicoline were estimated by measuring the thickness of the various retinal layers. In addition, immunohistochemistry was conducted to elucidate the expression of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH). Morphometric analysis of retinal damage in KA-injected eyes showed a significant cell loss in the inner nuclear layer (INL) and inner plexiform layer (IPL) of the retinas at the 1, 3 and 7 days after KA injection, but not in the outer nuclear layers (ONL). At 1 and 3 days after citicoline treatment, no significant changes were detected in the retinal thickness and immunoreactivities of ChAT and TH. The immunoreactivities of ChAT and TH had almost disappeared in the retina after 7 days of KA injection. However, prolonged citicoline treatment for 7 days significantly attenuated the reduction of retinal thickness and immunoreactivities of ChAT and TH. The present study suggests that treatment with citicoline has neuroprotective effect on the retinal damage due to KA-induced neurotoxicity. q 2005 Elsevier Ltd. All rights reserved. Keywords: choline acetyltransferase; tyrosine hydroxylase; kainic acid; citicoline; retina; rat
1. Introduction Glutamate is the main excitatory neurotransmitter in the CNS. In the retina, L-glutamate is highly concentrated in the photoreceptor, the bipolar and ganglion cell layer (Hara and Sukamoto, 1993; Berger et al., 1997; Wu and Maple, 1998). Intravitreal injection of kainic acid (KA), a structural analogue of L-glutamate, induces rapid and selective lesions in the inner retina of rats with the photoreceptor cell-sparing (Goto et al., 1981; McGeer, 1982); * Corresponding author. Dr Gyeong Jae Cho, Department of Anatomy and Neurobiology, College of Medicine, Gyeongsang National University, 92 Chilam-dong, Jinju, Kyungnam 660-751, South Korea. E-mail address: [email protected] (G.J. Cho).
0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.02.007
it is well known that glutamate receptor-related neurotoxicity can be induced by NMDA (N-methyl-D-aspartic acid) and KA (Ehrlich and Morgan, 1980; Morgan and Ingham, 1981; Siliprandi et al., 1992). Neuronal damage, followed by cell loss, also has been observed in the retinas exposed to relatively high concentrations of glutamate and glutamate analogues, such as activating NMDA receptors or KA/AMPA (kainic acid/a-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid) receptors (Lucas and Newhouse, 1957; Olney, 1969; Yazulla and Kleinschmidt, 1980; Hampton et al., 1981; Abrams et al., 1989; Osborne and Quack, 1992; Perez and Davanger, 1994; Secades and Frontera, 1995). An overstimulation of these receptors leads to increased intracellular Ca2C levels and constitutes one of the steps in the development of the excitotoxic retinal cell damage which is observed following ischemia (Bresnick, 1989).
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Citicoline, a naturally occurring endogenous nucleoside, also known as CDP-choline (cytidine-5-diphosphocholine), is one of the rate-limiting factor in the formation of phosphatidycholine (PtdCho), an essential phospholipid for the maintenance of intracellular and extracellular membranes in the brain (Kennedy and Weiss, 1956; Trovarelli et al., 1981). Citicoline crosses the blood–brain barrier as cytidine and choline, in rodents, but not in humans, where its circulating breakdown products are uridine and choline (Wurtman et al., 2000), which reach the brain and synthesize again the citicoline in the cytoplasm (Secades and Frontera, 1995). Citicoline and its hydrolytic products (cytidine and choline) play important roles in the generation of phospholipids, which are involved in membrane formation and repair; cytidine and choline also contribute to critical metabolic function such as the formation of nucleic acids, proteins and acetylcholine (Weiss, 1995). For these reasons, citicoline has been assayed as a therapeutic agent in a variety of central nervous system (CNS) injury models and neurodegenerative diseases. Further, a few clinical studies showed beneficial effects of citicoline on the function of the visual pathway in patients with retinal damage (Parisi et al., 1999). In the rat retina, amacrine cells mediate lateral interaction in the inner retina. They are involved in modifying receptive field properties of ganglion cells and temporally modulating signal transfer from the bipolar cells to the ganglion cells (MacNeil and Masland, 1998); the dopaminergic amacrine cells are associated with retinal adaptation, whereas the cholinergic amacrine cells are important for directional selectivity of a population of ganglion cells and contain ionotropic excitatory amino acid receptors. We investigated the retinal dopaminergic and cholinergic systems using tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in the rat retinas of KA-induced damage and showed neuroprotective action of citicoline in those cells by immunohistochemistry and morphometric analysis.
2. Materials and methods All animal experiments were conducted in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. 2.1. Animal models Male Sprague–Dawley rats (200–250 g) were maintained under controlled conditions of temperature (25 8C) and photocycle (12 hr on/12 hr off), and allowed free access to food and water. The animal number used in this experiment was 35 and divided into the three groups as control group (nZ5) and the two experimental groups (nZ15, each); KA injection group (1 day, 3 and 7 days, nZ5, each time point)
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and KA injection plus treatment with citicoline group (1 day, 3 and 7 days, nZ5, each time point). Rats were injected intraperitoneally with citicoline (500 mg kgK1, i.p.) twice (09:00 and 21:00) daily for 1, 3 and 7 days after KA-injection. For KA injection each animal was anesthetized with an intraperitoneal injection of a mixture of ketamine (30 mg kgK1; Yuhan Corporation, Seoul, Korea) and xylazine (2.5 mg kgK1; Bayer, Seoul, Korea) and placed in a stereotaxic frame. KA (sigma, USA) was dissolved in sterile normal saline. After topical application of 0.5% proparacaine hydrochloride (0.5% Alcaine; Alcon, USA) to the right eye, a single injection of 3 ml of 2 mM KA (total amount, 6 nmol) was done into the vitreous space with a microsyringe (Hamilton, Reno, NV) when the needle tip reached the midvitreous. These procedures were performed under an operating microscope. Gentamicin ophthalmic ointment was applied topically to the eye after cannulation of the vitreous space. 2.2. Tissue preparation The animals were sacrificed at 1, 3 or 7 days after KA injection and KA injection plus treatment with citicoline. The eyes were enucleated for morphologic and immunohistochemical (IHC) analysis. Immediately after enucleation, the eyes were cut open by a circular incision and the posterior eyecup was fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) for 6 hr at 4 8C, then rinsed in 0.1 M PB for 30 min at 4 8C. Tissues were rinsed in 30% sucrose in 0.1 M PB for 12 hr, frozen in embedding medium (O.C.T. Compound, Miles Inc., Elkhart, IN) in liquid nitrogen, and cryosectioned at a thickness of about 12 mm in a sagittal direction through the optic nerve using a cryostat (Leica 8400E; Leica, Tokyo, Japan). The sections were thaw-mounted on slides coated with 1% gelatin solution and stored at K70 8C until morphologic and immunohistochemical use. Some sections were stained with hematoxylin and eosin. Mounted sections were microscopically observed and photographed at the same magnification, and the images at a distance of approximately 0.8–1 mm from the optic nerve head of both control and experimental groups were measured. 2.3. Immunohistochemistry Retinal cryosections from control, KA injection and KA injection plus treatment with citicoline groups were rinsed in 0.02 M phosphate-buffered saline (PBS), pH 7.4, 0.5% Triton X-100 in 0.02 M PBS and again in 0.02 M PBS for 10 min each at room temperature before incubation in the primary antibody. They were then incubated with 3% normal goat or rabbit serum (Vector Labs, Burlingame, CA, USA), 5% bovine serum albumin (Gibco Labs, Grand Island, NY, USA), and 0.3% Triton X-100 in 0.02 M PBS for 30 min at room temperature. The following primary antibodies were used: polyclonal goat anti-choline
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Fig. 1. Representative light microscopic photographs of the rat retina under different experimental conditions. (A) Normal retina. Five well-organized retinal layers are seen; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer, GCL, ganglion cell layer. Histological features of transverse sections in the retina after 1 day (B), 3 days (D) and 7 days (F) of KA injection. In (B), the thickness of the retina is reduced due to the reduction of the inner retina (INL and IPL) as compared with the normal retina. In (D and F), the thickness of the retina is reduced due to the disappearance of the IPL and the INL. Histological features of transverse sections in the retina after 1 day (C), 3 days (E) and 7 days (G) of KA injection plus treatment with citicoline. Compared with those shown in (D and F), the thickness of the INL and IPL (E and G) increased. Calibration bar: 50 mm.
acetyltransferase (anti-ChAT) (1:100; Chemicon International Inc., Temecula, CA, USA), polyclonal rabbit anti-tyrosine hydroxylase (anti-TH) (1:200; Pel-Freez Biologicals, Rogers, Ark., USA), each in 3% normal goat or rabbit serum, 5% bovine serum albumin, and 0.3% Triton X-100 in 0.02 M PBS incubation solution. All incubations were performed overnight at 4 8C in a humidity-tight box, without agitation. To control non-specific binding of the antiserum, some tissue sections were incubated in antibodyfree incubation solution. The sections were then washed with PBS and Triton X-100 as described above. Secondary antibodies (affinity-purified biotinylated goat anti-rabbit or
rabbit anti-goat IgG (HCL), Vectastain ABC kit, Vector Labs) were diluted (1:200) in a incubation solution. The sections were exposed to secondary antibodies for 1.5 hr at room temperature, washed in PBS and Triton X-100 and incubated with avidin–biotin complex (Vectastain ABC kit, Vector Labs, diluted (1:50) in 0.02 M PBS) for 1.5 hr at room temperature. They were washed again in PBS and Triton X-100, rinsed in 0.05 M Tris–HCl buffer (pH 7.5) for 10 min at room temperature, and pre-incubated in 0.05% 3, 3 0 -diaminobenzidine tetrahydrochloride (DAB) in TB for 10 min with gentle rocking. Hydrogen peroxide was added to the same DAB solution to make a final concentration of
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0.01% H2O2. After 1–2 min, determined by the degree of staining using a light microscope (Olympus), the reaction was terminated with several washes in 0.002 M PBS. 2.4. Statistical analysis The results are the mean of results from five adjacent areas within 1 mm of the optic nerve. All measurements were analyzed using an image analysis program (Soft Imaging system GmbH, Germany). For accurate analyses of the decreased TH- and ChAT-positive amacrine cells, we selected sectioned retinas with intact morphology to both ends of the ciliary bodies, centering on the optic nerve. THand ChAT-positive amacrine cells in the INL and/or IPL were then counted per retina, and normal and experimental groups were compared. In particular, the number of ChATand TH-like immunoreactive amacrine cells in the GCL and INL were counted on five adjacent retinal sections of individual animals. Values were given as meanGS.E. Statistical significance of difference between groups was performed using an unpaired Student’s t-test.
3. Results 3.1. Morphometric analysis for neuroprotective effect of citicoline Intravitreal injection of KA caused neuronal damage to retinal tissue. In the KA-treated retina, the entire retinal thickness decreased gradually compared with the control retinas. Specifically, the thickness of the retina after 7 days of KA injection was reduced to about 43% of the control retinas (Figs. 1 and 2). Fig. 2 shows the changes in the thickness of the major retinal layers in controls and KAtreated rats at various time points after KA injection. The effects on the individual retinal cell layers were different with respect to the time-course and extent of thickness reduction (Fig. 2). The thickness of the inner plexiform layer (IPL) started to decrease in the retina after 1 day of KA injection (Fig. 1B). In the retina after 3 days of KA injection, the thickness of the IPL and inner nuclear layers (INL) was gradually diminished (Fig. 1D). At 7 days, there was a significant decrease in overall retinal thickness, with a marked thinning of the IPL and the INL, whereas the thickness of outer retinal layers (ONL) was changed only slightly (Fig. 1F). The protective effects of citicoline on the KA-induced neuronal damage were examined by measuring the thickness of the ONL, INL and IPL. In the citicoline treated retina, after 3 and 7 days of KA injection, there was a significantly attenuated reduction in the thickness of the INL and IPL caused by KA treatment (Figs. 1 and 2). The retinal layers of the citicoline-treated retina showed relatively wellmaintained structures compared with untreated retinas.
Fig. 2. Morphometric analysis of KA-induced changes in the thickness of the outer nuclear layer (ONL, A), inner nuclear layer (INL, B) and inner plexiform layer (IPL, C) of the untreated and citicoline-treated rat retinas. Values were measured in retinal sections shown in Fig. 1. The degree of reduction in the thickness of the retina was quantified by the thickness (mm) of retinal layers such as the ONL, INL and IPL at a distance of 1.0–1.5 mm from optic disc. The thickness of the retinal layers gradually diminished in the inner retina (INL, *P!0.05 and IPL, **P!0.001) of citicoline treated group as compared with the normal retina after KA injection (B and C); the reduction was significantly attenuated when animals were treated with citicoline. The results are shown as meanGS.E. Statistical significances were calculated by Student’s t-test between control and experimental retinas (*P!0.05).
3.2. Effect of citicoline on ChAT immunoreactivity in the retina after KA injection The effect of citicoline on ChAT expression in the retina at different time points after KA injection is shown by
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Fig. 3. Photomicrographs of choline acetyltransferase (ChAT) immunoreactivity of controls (A); after 1 day (B), 3 days (D) and 7 days (F) of KA injection and after 1 day (C), 3 days (E) and 7 days (G) of KA injection plus treatment with citicoline. In the control retinas (A), ChAT immunoreactivity appeared as two clearly defined bands (arrow) in the inner plexiform layer with cell bodies (arrowheads) in the INL and GCL. ChAT immunoreactivity almost disappeared after 7 days of KA-injection (F). Prolonged citicoline treatment prevented the reduction in the ChAT immunoreactivity induced by KA injection (G, arrows). Calibration bar: 50 mm.
immunohistochemical analysis (Fig. 3). In the control retina, ChAT immunoreactive cells were associated with two types of certain amacrine cells located in both the INL and the GCL, and two clear laminae (arrow) in the IPL (Fig. 3A). ChAT immunoreactivity started to decrease after 1 day of KA injection (Fig. 3B). The immunoreactivity of ChAT in retinas subjected to KA-injection showed clear changes when compared with the control retinas. The normal immunoreactivity in the IPL was reduced in intensity and often appeared as a single band (Fig. 3D). The retina after 7 days of KA injection displayed an almost complete disappearance of ChAT immunoreactivity (Fig. 3F), but the ChAT immunoreactivity was significantly protected by a treatment with citicoline for 7 days (Fig. 3G).
The number of ChAT-positive cell in the inner nuclear layer (INL) and ganglion cell layer (GCL) were counted. The number of ChAT-positive cells gradually decreased compared to the control retinas as time passes after KA injection (Fig. 4; **P!0.001, respectively; nZ5). 3.3. Effect of citicoline on tyrosine hydroxylase immunoreactivity in the retina after KA injection Tyrosine hydroxylase (TH) immunoreactivities in control, citicoline-treated and non-treated rat retinas are shown in Fig. 5. The immunoreactive amacrine cells consisted of two types, a small and a large type and the cell somata were located in the outer part of the INL with branches oriented
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Fig. 4. Cell counts of ChAT-positive cells. The number of ChAT-positive cell in the inner nuclear layer (INL) and ganglion cell layer (GCL) were counted. The number of ChAT-positive cells gradually decreased compared to the control retinas as time passes after KA injection. The retina showed an almost disappearance of ChAT-positive cells after 7 days of KA injection. Prolonged citicoline treatment for 7 days significantly protected the reduction in the number of ChAT-positive cells. Statistical significances were calculated by Student’s t-test between control and experimental retinas (**P!0.001).
mainly towards the inner layers in the retina. The processes emerging from these cells ramified mainly in the outer part (sublamina 1, small arrow) of the IPL (Fig. 5A). The retinal TH immunoreactive cells were sparsely distributed throughout the retinas after 1 and 3 days of KA injection (Fig. 5B and D). The retina showed an almost complete disappearance of TH immunoreactivities after 7 days of KA injection (Fig. 5F). In the retinas after 1 and 3 days of citicolinetreatment, the immunoreactivities of TH were not changed significantly compared with KA-treated retinas (Fig. 5C and E). However, prolonged citicoline treatment for 7 days prevented the entire disappearance of TH immunoreactivities induced by KA injection (Fig. 5G). We counted all large or small TH-positive amacrine cells in the INL–IPL margin per retina and carried out a comparative analysis between the control and experimental groups. TH-amacrine cells gradually reduced in the KA injection groups compared to controls, showing a 28, 53 and 90% decrease at 1, 3 and 7 days after KA injection, respectively (Fig. 6; **P!0.001, respectively; nZ5).
4. Discussion The present study demonstrated that citicoline has a neuroprotective effect on experimental models of KAinduced retinal damage. We found that citicoline treatment twice daily for 7 days prevented the loss of retinal ChAT and TH immunoreactivities, as well as the thinning of the inner retinal layers resulting from KA-injection. Injection of KA to the rat eye caused a destruction of the retinal neurons, which was counteracted by continuous treatment with
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citicoline for 7 days. This study clearly shows the change of inner retinal thickness in the retina of KA-induced damage model. The retinal cell damage occurred in the inner retina up to 1 day after KA injection, whereas cells in the ONL were damaged later. Morphological evaluation of transverse sections of retina with KA injection demonstrated a dose-dependent loss of cells in the INL and a reduction in the thickness of the IPL (Morgan and Ingham, 1981); low doses (6–20 nmol) of kainic acid reduce the content of the amacrine and horizontal cell markers acetylcholine and gamma-amino-butyric acid (GABA); higher doses of kainic acid lead to disappearance of both the outer and inner plexiform layers. The KA-dose in this study was 6 nmol, comparable to low dose of the previous study and our results are in good accordance with the finding of this report where amacrine acetylcholine was reduced compared to control. Clinical reports indicate that citicoline may have a therapeutic effect in patients with glaucoma. Some researchers reported that citicoline ameliorated ganglion cells death in vitro (Oshitari et al., 2002). However, the precise effect of citicoline on damaged retinal ganglion cells (RGCs) remains to be explained. Previous studies have shown that the KA-induced retinal ganglion cell death appears to be mediated via NMDA receptors, suggesting that KA toxicity is mediated by release of glutamate, which in turn activates NMDA receptors. KA increases the proportion of retinal ganglion cells that die, but the toxicity (due to both KA and the endogenous toxin) is totally prevented by 2-amino-5-phosphonovalerate (APV), a specific NMDA receptor antagonist (Sucher et al., 1991). Although the exact mechanisms by which citicoline produces its neuroprotective effects are unknown, it has been shown to reduce neuronal degeneration by inhibiting the apoptotic pathway induced by glutamate (Mir et al., 2003). Citicoline may have the neuroprotective action in glutamate-mediated cell death. ChAT is a reliable marker for cholinergic amacrine cells, which are located on either side of the IPL, in the INL and GCL of the rat retina. The dendrites of the ChAT immunoreactive cells are located as two distinct strata in the IPL. The protective effect of citicoline on the rat retina was demonstrated by an analysis of ChAT immunoreactive cells. We found that by 7 days after KA injection there was an almost complete disappearance of ChAT-positive cells. It is known that ChAT positive amacrine cells in the retina are highly vulnerable to intraocular injection of neurotoxins (Gomez-Ramos et al., 1985). So it is tempting to speculate that the vulnerability of ChAT-positive cells in the KAtreated retina may be due to the presence of kainate/AMPA and NMDA receptors on those neurons (Brandsta¨tter et al., 1994; Osbone et al., 1995; Peng et al., 1995). It is likely that the disappearance of ChAT immunoreactivity might be due to an excessive influx of calcium from an overactivation of ionotropic glutamate receptors (Louzada-Junior et al., 1992). In the present study, our results demonstrated that
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Fig. 5. Photomicrographs of tyrosine hydroxylase (TH) immunoreactivity of controls (A); after 1 day (B), 3 days (D) and 7 days (F) of KA injection and after 1 day (C), 3 days (E) and 7 days (G) of KA injection plus treatment with citicoline. In the control retinas (A), the immunoreactivity amacrine cell consisted of two types, a small (open arrow) and a large type (solid arrow). The retina showed an almost complete disappearance of TH immunoreactivity after 7 days of KA injection (F). This loss was significantly protected by prolonged citicoline-treatment, showing apparent TH immunoreactivity (G, arrowhead). Calibration bar: 50 mm.
citicoline-treatment counteracted the reduction of the ChAT immunoreactivity caused by kainic acid injection, showing that citicoline can act as a neuroprotective agent against kainic acid-induced retinal damage. Dopaminergic cells contain tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and these cells react with antibody specific for TH. In the rat retina, the majority of dopaminergic cells are located in the innermost sublayer of the INL. It was previously reported that there are two types of TH immunoreactive cells in the adult rat retina (Wu and Cepko, 1993; Martin-Martinelli et al., 1994). Type I cells are large, located in the INL and react strongly with anti-TH. Their dendrites ramify at the junction between the INL and the IPL. Type II cells are small and only slightly reactive with anti-TH. They are
located more deeply within the INL (Martin-Martinelli et al., 1989; Martin-Martinelli et al., 1994; Yoles and Schwartz, 1998). The results are consistent with these reports. Intraocular injection of KA, a powerful glutamate receptor agonist, induced a significant decrement in the specific activities of tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and degeneration of cells in the inner nuclear layer of the retina (Schwarcz and Coyle, 1977). Citicoline was shown to stimulate tyrosine hydroxylase activity and dopamine release (Secades and Frontera, 1995). Thus, neuroprotection following citicoline treatment in KA-induced retinal damage may be the consequence of stimulating in cholinergic and dopaminergic system. In conclusion, the results demonstrate that the citicoline can spare cells from KA-induced retinal damage, suggesting
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Fig. 6. Cell counts of TH-positive cells. TH-positive amacrine cells in the INL were counted. The number of TH-positive cells gradually decreased compared to the control retinas as time passes after KA injection. The retina showed an almost disappearance of TH-positive cells after 7 days of KA injection. Prolonged citicoline treatment for 7 days significantly protected the reduction in the number of TH-positive cells. Statistical significances were calculated by Student’s t-test between control and experimental retinas (**P!0.001).
that citicoline is an efficient drug for the treatment of experimental neurodegenerative retina wherein cholinergic and dopaminergic neurons are related.
Acknowledgements This Research was supported by a grant (M103KV010020 03K2201 02010) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of Republic of Korea and partially supported by a grant of the BK 21 Project of Ministry of Education.
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