β-Amyloid protein-induced Alzheimer's disease animal model

the concomitant loss of the cholinergic marker enzyme, choline acetyltransferase (CHAT), in the cerebral cortex [21]. However, direct evidence, that fl-amyloid protein is related to the impairment of learning and memory, has not been obtained. We now show that memory impairment and neuronal dysfunction of cholinergic neurons were produced after continuous infusion of fl-amyloid protein into the cerebral ventricles in adult rats. These results in this study, suggest that the deposition of flamyloid protein in the brain is related to the impairment of learning and the cholinergic neuronal degeneration, and that fl-amyloid protein-treated rats could be used as an animal model for AD. Male Kbl Wistar rats (Kitayama Laboratories Co., Kyoto), weighing 280 320 g at the beginning of the experiments, were used. They were housed in groups of two or three in a temperature- and light-controlled room (23°C; 12-h light cycle starting at 09.00 a.m.). The rats had free access to food and water, except during the experiments. The synthesized human fl-amyloid protein(1M0) was kindly provided by Shionogi Co. Ltd. (Osaka, Japan). The fl-amyloid protein was dissolved in 35% acetonitrile/0.1% trifluoacetic acid (TFA). Continuous infusion of the fl-amyloid protein (0, 3, 30, 300 pmol/ day) was maintained for two weeks by attachment of a cannula to a modified mini-osmotic pump (Alzet 2002;
ELΒιβλιοθήκη BaiduEVIER
Neuroscience Letters 170 (1994) 63-66
N[UROSCIENCE [ETT[R$
fl-Amyloid protein-induced Alzheimer's disease animal model
Atsumi Nitta, Akio Itoh, Takaaki Hasegawa, Toshitaka Nabeshima*
Key words: fl-Amyloid protein: Alzheimer's disease; Memory; Choline acetyltransferase: Water maze: Rat
Alzheimer's disease (AD) is characterized by the presence of senile plaques and neurofibrillary tangles accompanied by synaptic and neuronal loss. The core of the plaque consists offl-amyloid protein [1,9] and other proteins [17]. The extent of fl-amyloid protein deposition correlates with the degree of neuronal damage, cognitive impairment, and memory loss [8,20]. Although this protein has been well characterized biochemically, its primary biological function and role in the pathogenesis of AD are unknown [12]. Yankner et al. have reported that fl-amyloid protein in vitro has both neurotrophic and neurotoxic effects that depend on neuronal age and the concentration of flamyloid protein [22]. Additionally, they have shown that fragments offl-amyloid, as well as the complete peptide sequence offl-amyloid(l~0), were neurotoxic to the hippocampus cells in primary culture and that direct injection of fl-amyloid into the hippocampus of rats resulted in a pattern of characteristic changes of AD; fl-amyloid induces Alz-50-immunoreactive protein in rat cerebral cortex that was very similar to the protein induced in human cerebral cortex from patients with AD [7]. In AD patients, learning and memory are impaired by
64
A, ,Villa cl JL i Ncttro.w'ieHc~ ~ l.ctter~' 1,70
1994~ 63 fJ6
Fig. 1. l'hc dcpo~ltol~ oI mlmuno~'cactixity \~ith/~-tllll~]Old t~rotcm
Alza, CA) [13]. Each group consisted of seven rats. The cannula was implanted into the left ventricle (A -0.3, L 1.1, V 3.6) on the day 1. The water maze task was carried out on the day 9-13 after the start of infusion. After the behavioral experiments, four rats of each group were decapitated for ChAT activity assay. Three rats were used for histochemical study. In the histochemical study, rats were anesthetized and killed by transaortic perfusion-fixation with cold saline followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The brains were removed, postfixed for 12 h in the same fixative. The brains were left in 10% sucrose in phosphate-buffered saline (PBS) for 4 h, a 15% one for 4 11, and a 20% one for 12 h at 4°C. Frozen brains were cut at 20/.tm with a cryostat and collected the areas around the ventricles. A mouse anti-human fl-amyloid protein(8-17) monoclonal antibody (Dakopatts A/C, Glostrup, Denmark) was used at a 1: t00 dilution. The tissues were incubated overnight at 4°C with the primary antibody. Immunolabeled sections were processed and developed with diaminobenzidine using Vectastain kit (Vector Laboratories, CA). Water maze task was carried out as reported previously [11,15]. The animal was trained for 90 s. When it failed to get to the hidden platform, the training was terminated and a maximum score of 90s was assigned. Training was carried out twice a day for 5 days. Measurement for ChAT activity was carried out as reported previously [5,14]. The data from the maze task were analyzed by a repeated-measure analysis of variance and Tukey's test. ChAT activity data were analyzed using one-way analysis of variance and Tukey's test.
*Corresponding author. Fax: (81) (52) 733-9415. 0304-3940/94/$7.00 (~ 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00097-T
Abstraet
To investigate the toxicity of fl-amyloid protein which consisted of senile plaques of Alzheimer's disease (AD), this was infused into cerebral ventricle for 14 days by using mini-osmotic pump. The performance of the water maze task in fl-amyloid protein-treated rats was impaired. Choline acetyltransferase activity significantly decreased in the frontal cortex and hippocampus. These results suggest that the deposition of fl-amyloid protein in the brain is related to the impairment of learning and cholinergic neuronal degeneration, and that fl-amyloid protein-treated rats could be used as an animal model for AD.
D~Tmrtment ~/ Neuropo'chopharmacology and Hospital Pharmacy, Nagoya UniversiO' School o/" Medicine, Tsurumai-cho 65, Showa-ku. Nagoya 466, Japan
Received 24 December 1993; Revised version received 24 January 1994; Accepted 24 January 1994