Toxicological Sciences 66, 253-260 (2002)
Copyright © 2002 by the Society of Toxicology
NEUROTOXICOLOGY |
Aluminum Chloride Induces Retinal Changes in the Rat
Department of Ophthalmology and Visual Sciences, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki-shi 852-8501, Japan
Received August 17, 2001; accepted November 16, 2001
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We studied rat retinal changes due to aluminum (Al) toxicosis with a transmission electron microscope (TEM) and an energy dispersive X-ray analyzer (EDXA). Normal 4-week-old Wistar Kyoto rats were divided randomly into Al toxicosis and control groups. The Al toxicosis group was injected ip with 0.3 ml of 4% aluminum chloride (AlCl3) per day every day for 16 weeks. The retina was examined with a TEM and EDXA at 8, 12, and 16 weeks after starting injections with AlCl3. There was a statistically significant increase in the serum Al concentration in the Al toxicosis group (p < 0.001). We observed prominent pathologic changes at 16 weeks after the first injections. Thin retinal pigment epithelium (RPE), and disappearance of the photoreceptor outer and inner segments and nuclei were observed. There were high-density irregular granules in the outer and inner plexiform layers and in the inner nuclear layer. We found dense granules in the cells, which remained between the RPE and the inner nuclear layer. EDXA detected Al in the high-density irregular granules in these areas. Al injected ip caused accumulation of Al in the rat retina and the destruction of photoreceptor cells. These findings indicate that Al is toxic to the retina.
Key Words: aluminum; rat; retina; electron microscopy; energy dispersive X-ray analyzer.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Aluminum (Al) is one of the most abundant elements in nature. Al was recognized as a neurotoxin 100 years ago (Lukiw, 1997
), but it was not until 1921 that the specific effects of Al on the human central nervous system (CNS) such as memory loss and impaired coordination were first documented in a medical journal (Lukiw, 1997; Spofforth, 1921). Al is clinically well known to be toxic to the CNS, skeletal system, and hematopoietic system in humans. Exposure to Al is common by oral intake, inhalation, transdermal introduction, or via potential administration through hemodialysis. There have been many neurological and pathological studies of potential Al toxicity (Wilhelm et al., 1990; Yokel and McNamara, 2001). The injection of Al compounds into animal brains has been found to produce neurofibrillary degeneration (NFD) in the CNS similar to that observed in Alzheimer's disease (AD; Crapper, et al., 1980; De Boni et al., 1974). The retina and optic nerve are derivatives of the forebrain; consequently their morphology and physiology are very much like that of the brain (Hogan et al., 1971). Since it has an affinity for the CNS, Al may be deposited in ocular tissue. However, that possibility has not been studied in the context of the above-mentioned neurological disorders. Since the retina is part of the CNS, we hypothesized that it might have an affinity for Al and therefore might be easily damaged by exposure to high levels of Al. In this report, we examined the ultrastructure and Al deposition in the retina of rats injected ip with Al; exposure lasted for up to 16 weeks. The retina was then analyzed by energy dispersive X-ray analyzer (EDXA) for Al deposition.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normal 4-week-old Wistar Kyoto rats were divided randomly into Al-treated (n = 20) and control groups (n = 10). The animals were maintained at a constant temperature of 21°C with a 12-h light/dark cycle in the Laboratory Animal Center for Biomedical Research, Nagasaki University School of Medicine. The rats were treated in accordance with the ARVO resolution on animal research. The Al toxicosis group was injected ip with 0.3 ml of 4% aluminum chloride (AlCl3) every day for up to 16 weeks. The rats were killed (at least 5 rats in each group) on the first day of the 8th, 12th, and 16th week after the start of administration of AlCl3. The posterior pole of the retina of rats treated with Al was examined on the day following the last injection. Eyes were enucleated under pentobarbital sodium (Dainabor, USA) anesthesia, and blood was simultaneously drawn from the aorta 8, 12, and 16 weeks after the start of the experiment. The measurement of serum aluminum levels was performed by an atomic absorption spectrophotometer. Age-equivalent normal Wistar Kyoto rats were used as controls and were treated with physiological sodium solution instead of aluminum chloride.
TEM procedure.
Enucleated eyes were fixed in 4% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) for 1 h and were post-fixed in 1% osmium tetroxide in veronal acetate buffer for 1 h, after overnight washing with 0.05 M cacodylate buffer (pH 7.4) containing 0.44 M sucrose. The fixed materials were dehydrated with a series of ethanol concentrations and embedded in Luveak 812. Ultrathin sections were cut with a Porter-Blum MT2 microtome, stained with uranyl acetate and lead citrate, and examined with a Hitachi H-300 TEM (Tokyo, Japan).
EDXA procedure.
Ultrathin sections were cut with a Porter-Blum MT2 microtome and examined without staining with a JEOL-1210 TEM (Tokyo, Japan) and EDXA DX-4 (Philips, The Netherlands). The analytical conditions were as follows: magnification x10,000 or x4000; accelerating voltage, 100 KeV; sample current, 60 µA; and time, 200 s.
Statistical analysis.
The photoreceptor cell nuclei were counted in a 30 mm2 area on electron micrographs taken at magnification x1200. All data were analyzed by unpaired Student's t-test with least significant difference. Differences were considered significant at p < 0.05. All values represent mean ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The serum Al level increased with the dose of Al administered; in the controls it was always less than 10 µg/l, the limit of sensitivity (Table 1
). The difference between Al-treated and control groups was significant (p < 0.001) (Table 1). Al-treated rats showed no abnormal changes in weight gain or body hair, but the entire body of rats treated for 16 weeks showed severe torsive activity.
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In rats treated with AlCl3 for 8 and 12 weeks, the photoreceptor outer nuclei did not decrease (Table 2
), but Müller cells in the outer nuclear layer were vacuolated and some had disappeared altogether (Figs. 1–4). EDXA failed to detect Al in any of the intracytoplasmic organelles, including lysosomes, mitochondria, dense bodies, nucleus, photoreceptor outer segment discs, and abnormal granules suspected of containing Al-related substances.
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Rats treated with AlCl3 for 16 weeks showed prominent pathologic changes, i.e., a thin retinal pigment epithelium (RPE) and disappearance of the outer and inner segments and nuclei of photoreceptors (Fig. 5
). The retina between the RPE and the inner nuclear layer was almost completely destroyed and contained unidentifiable cells that had no cytoplasmic membrane; debris of intracytoplasmic organelles and dense irregular granules were also observed. Pyknotic nuclei were seen in unidentified cells in the inner nuclear layer, but no apoptotic nuclei. The granules were also seen in dendrites and/or neurons in the inner and outer plexiform layers due to the damaged retinal structure. Very dense irregular granules were seen in the nuclei and the cytoplasm of unidentified cells between the inner nuclear layer and the RPE (Fig. 6). These granules appeared in intracytoplasmic debris, in the nucleus, in the cytoplasmic matrix (Fig. 7), and in the RPE. The RPE cells were dark and contained numerous granules (Fig. 8A).
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EDXA detected Al at the dense irregular granules in the unidentified cells between the inner nuclear layers and in the RPE cells (Fig. 8
), between the RPE and neurons and dendrites in the inner plexiform layer, and in the outer plexiform layer as well. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study showed high concentrations of Al in the serum of rats injected ip with 5 or more mg Al/kg daily for 8–16 weeks, and EDXA showed Al in their degenerated retinas.
Experimental Al intoxication has certain silent characteristics; namely, it is hard to induce Al intoxication by specific doses and therefore it takes a long time to induce Al intoxication. In addition, Al intoxication occurs suddenly after long-term administration of Al, and the effects of Al exposure might be indirect rather than direct, due to Al accumulation (Swegert et al., 1999). There have been no reports of abnormal retinal function due to Al intoxication. In the present study, we used AlCl3 and the dose was determined according to that used in previous reports (Ebina et al., 1984; Uemura, 1984). The rats that were administered 0.3 ml of 4% AlCl3 daily first showed retinal changes at the 8th week of the experiment, and they showed very slight changes at the12th week. Prominent changes in the retina (severe degeneration and the disappearance of photoreceptor cells) were first observed at the 16th week; Al was also detected by EDXA in dense granules in the inner nuclear layer nuclei, inner plexiform layer, dendrites and/or neurons of the outer plexiform layer. These progressing changes correlate with the serum Al level.
Thus, long-term exposure with a large quantity of Al may be necessary for Al intoxication. The total dose of Al administered during the present experiment is unusually high, and such excessive amounts of Al do not enter the human body. In fact, Al intoxication occurs in hemodialysis patients after long-term hemodialysis (Lin et al., 1996). Although Al was first demonstrated in the retina of rats treated with AlCl3 for 16 weeks, there is a possibility that the retina already contained excessive Al in rats treated for 8 or 12 weeks, since the threshold sensitivity of EDXA is too low to detect the element in the cytoplasm without deposits of Al. Rats treated for 16 weeks showed retinal degeneration with deposits of Al in the retina. In addition, cell nuclei were pyknotic but not apoptotic. These findings suggest that Al may combine with chromatin in the nuclei and in neurons and/or dendrites of the outer and inner plexiform layers. Histochemical and subcellular fragmentation studies on intracellular binding sites of Al show that Al concentration specifically increases in the large euchromatic neocortical neuronal chromatins (De Boni et al., 1974; Lukiw et al., 1992, 1987; McLachlan, 1986; Wen and Wisniewski, 1985). Experimental aluminum encephalopathy (EAE) shows that Al lactate or Al chloride injected into an Al-susceptible animal induces a rapid accumulation of Al in the nuclear structure of the glia and large neurons (Crapper et al., 1980; Crapper-McLachlan and De Boni, 1980; Wen and Wisniewski, 1985). In EAE neuronal tissue, a high concentration of Al is found in glial or neuronal lysosomes (Schuurmans-Stekhoven et al., 1990). The photoreceptor cells are assumed to be the most sensitive to Al toxicity, because they were the most severely damaged. In hemodialysis patients, high Al levels have been observed in the periosteum and epiphyseal cartilage. Certain brain lesions such as AD amyloid structures and other encephalopathies contain Al and are related to Al deposition. Similarly, in the present experiments, the Al deposits were found not only in the nucleus but also in the cytoplasmic matrix. The present study may offer some direction to the study of Al intoxication and other diseases.
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NOTES |
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1 To whom correspondence should be addressed. Fax: +81-95-849-7347. E-mail: d398048a{at}stcc.nagasaki-u.ac.jp.
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