Research ReportCerebral ischemia combined with β-amyloid impairs spatial memory in the eight-arm radial maze task in rats
Introduction
Senile plaques, composed of aggregations of β-amyloid (Aβ), are a pathological hallmark of the brain in Alzheimer's disease. Aβ is a 38- to 43-amino-acid peptide produced from amyloid precursor protein (APP) by proteolytic processing. Mutations in the APP (Cai et al., 1993), presenilin 1 (PS1) (Palacino et al., 2000, Palacino et al., 2001) and presenilin 2 (PS2) (Vito et al., 1996, Wolozin et al., 1996) genes, that are considered to cause or promote the onset of familial Alzheimer's disease (FAD), all lead to abnormal accumulation of Aβ, confirming a significant role for Aβ in Alzheimer's disease. Yankner et al. (1990) studied the pathogenic role of Aβ in Alzheimer's disease and demonstrated that Aβ itself is neurotoxic to cultured hippocampal neurons. We also reported that treatment with fibrillar Aβ induces prominent neuronal cell death and expression of cell-death-related early genes in primary cortical neuronal cultures (Iwasaki et al., 1996a, Iwasaki et al., 1996b). Similar findings were obtained in primary cultures of hippocampal neurons with cell death exhibiting morphological features of apoptosis (Egashira et al., 2002). Although the mechanism underlying Aβ neurotoxicity remains controversial, these findings provide strong evidence that Aβ plays a pivotal role in the pathogenesis of Alzheimer's disease.
While recent progress in Alzheimer's research has begun to unravel the genetic mechanisms underlying the disease, there have been few in vivo animal models suitable for therapeutic drug development. Molecular biological approaches, however, have enabled development of transgenic mice expressing FAD-linked mutant genes. Transgenic mice expressing a mutant APP gene develop senile plaques associated with excessive accumulation of Aβ (Hsiao et al., 1996) and mice expressing a mutant tau gene exhibit neurofibrillary changes caused by hyperphosphorylation of tau protein (Tatebayashi et al., 2002). These transgenic models closely reflect neuropathological features of Alzheimer's disease and have significantly contributed to research. However, the production of transgenic animals is technically demanding and costly and not suited for drug development that requires a large number of animals at any one time. Thus, a simpler and less costly method of producing animal models needs to be developed. Although previous behavioral pharmacological studies utilized rat models produced by scopolamine treatment or destruction of the basal ganglia, these treatments failed to induce neuronal apoptosis or Aβ accumulation seen in Alzheimer's disease brain and are therefore not ideal for Alzheimer's research. Hence, our group created a rat model of Alzheimer's disease by direct intracerebroventricular (i.c.v.) administration of Aβ. As Aβ is metabolized and eliminated relatively rapidly in the brain (Banks et al., 2003), Aβ was administered daily. I.C.V. administration of Aβ was first performed by the Nabeshima group (Nitta et al., 1994), however, we combined i.c.v. administration of Aβ with cerebral ischemia for the following reasons: (1) clinical reports indicate that Alzheimer's patients with a history of cerebrovascular disease have a more rapid progression of dementia (Snowdon et al., 1997); and (2) hypoxia treatment enhances Aβ-induced apoptosis in cultured hippocampal neurons (Egashira et al., 2002). Our group previously demonstrated that transient cerebral ischemia for 10 min causes impaired spatial memory in the eight-arm radial maze task 24 h after reperfusion, but this impairment can be easily reversed by repeated training and animals exhibit no signs of impairment after 7 days. A 10-min period of cerebral ischemia does not induce hippocampal apoptosis. Therefore, whether i.c.v. microinjection of Aβ combined with transient cerebral ischemia (10 min) in rats causes spatial memory impairment in the eight-arm radial maze task, and whether this treatment induces apoptotic changes in the CA1 region of the hippocampus was examined. Acetylcholine (ACh) release was also measured by microdialysis in the dorsal hippocampus of freely moving rats, in order to determine whether cholinergic dysfunction seen in most Alzheimer's patients was observed in this rat model. The validity of this rat model was also evaluated by analyzing the effects of donepezil, a drug widely used for the treatment of Alzheimer's disease (Shintani and Uchida, 1997).
Section snippets
Effects of cerebral ischemia and Aβ on spatial memory
In the eight-arm radial maze task, no significant changes in performance were observed either with ischemia [correct choices (CC), 7.3 ± 0.4; error choices (EC), 1.0 ± 0.5] or Aβ treatment alone (CC, 7.0 ± 0.2; EC, 2.3 ± 0.8), compared to the sham group (CC, 7.6 ± 0.2; EC, 0.7 ± 0.3) (Fig. 1). However, the combination of ischemia and Aβ significantly decreased the number of CC (6.2 ± 0.2) and significantly increased the number of EC (4.5 ± 0.9) compared to the sham group (P < 0.01 for CC and
Discussion
Aβ is produced by proteolytic processing of APP. Normally, Aβ is enzymatically degraded and metabolized, but when specific forms of Aβ peptide with 40 (Aβ1–40) or 42 amino acids (Aβ1–42) are created for unknown reasons, they are prone to adopt a β-sheet conformation and readily aggregate in the brain as amyloid fibrils. Accumulating evidence suggests that the β-sheet conformation of Aβ exerts neurotoxic effects. Since Yankner et al. reported Aβ-induced neuronal cell death in cultured neurons in
Animals
Male Wistar rats, aged 7 weeks and weighing 200–250 g, were obtained from Kyudo (Saga, Japan) and were housed in groups of four to five per cage in a room with the temperature controlled at 23 ± 2 °C, relative humidity of 60 ± 10% and the lights on from 7:00 to 19:00 h. The animals were restricted in their food intake (10–12 g each day, CE-2; Clea Japan, Tokyo, Japan), and body weight of each rat was maintained at ∼ 80% of the freely feeding level. All animals had free access to drinking water
Acknowledgments
We thank Eisai for the generous supply of donepezil. This study was supported by the Advanced Material Institute, University of Fukuoka and a Grant-in-aid for Scientific Research #13672407 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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