Intracerebroventricular Injection of Amyloid-β Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Summary

The amyloid-β (Aβ)-injected animal model enables the administration of a defined quantity and species of Aβ fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of Aβ without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Abstract

Amyloid-β (Aβ) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of Aβ oligomers and plaques is observed. Such Aβ abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of Aβ levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of Aβ is necessary in many cases. In this protocol, the steps of the experiment with an Aβ-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the Aβ into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of Aβ. By mimicking certain aspects of AD with a designated injection of Aβ, researchers can bypass the aging process and focus on the downstream pathology of Aβ abnormalities.

Introduction

Given that amyloid-β (Aβ) is a pathological hallmark of Alzheimer's disease (AD), the development of AD animal models has focused on neural overexpression of Aβ. Because mutations in amyloid precursor protein (APP) or presenilin (PS) lead to disturbance of Aβ equilibrium and ultimately to the pathogenesis of familial AD¹, mouse models involving APP or PS gene mutations have been generally accepted. Among the wide range of transgenic mice, prototypical mouse models include the following: TG2576, PDAPP, APP/PS1 and APP23. In the brain, these mice generally exhibit Aβ aggregation and eventually senile plaques; plaque formation is f....

Protocol

All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and with the Animal Care and Use Committee of the Institutional Animal Care and Use Committee of KIST (Seoul, Korea).

1. Animal Preparation

1. Prepare a group of 7-week-old male ICR mice (or C57BL/6).
2. Let the mice go through an acclimation process for 3-7 days after transport to restore homeostasis. In general, place a group of 4-5 mice in each cage and maintain them at 22-23 °C and 40% humidity, with a 12/12 hr light/dark cycle. Provide water and food ad li....

Results

This section illustrates examples of the results that can be obtained by confirmation of Aβ aggregation and Y-maze assessment of memory deficits. Using the full-length Aβ(1-42) peptide of 42 amino acids, mixture of Aβ monomers, oligomers, and fibrils (Figure 3) was produced. Through the HFIP-induced monomerization step, relatively homogeneous monomers (Lane B) were obtained. After the 7 day incubation, diverse sizes of Aβ aggregates (Lane C) developed. Trimers and tetrame.......

Discussion

The most important step in this protocol is the ICV injection of Aβ. This protocol is designed to inject Aβ into the ICV region of mice without stereotactic instruments^11,12. Before starting an experiment, a preliminary period of practice injections with a blue dye instead of Aβ should take place to achieve sufficient dexterity. Immediately after the injection, it is necessary to check whether the pigment injection was performed properly. Carefully take out the brain and check whether the approp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ICR mouse	Orientbio		male, 6-8 weeks, 27-29 g of body weight
C57BL/6 mouse	Orientbio		male, 6-8 weeks, 21-23 g of body weight
Amyloid-beta1-42	in house synthesis		stock concentration: 1 mM/DMSO, injected concentration: 100 μM/10% DMSO and 90% PBS
ICV injection syringe (26s gauge)	Hamilton	80308
Evans blue dye (EBD)	abcamBIochemicals	ab120869	1% EBD in PBS
DMSO	Sigma	D2650
PBS	gibco	10010-023
Gradi-GelTM II Gradient PAGE Analysis Kit	ELPiS Biotech	EBS-1056	15% Gel
Precision Plus ProteinTM Dual Xtra Standards	Bio-Rad	161-0377
Silver-Staining Kit	GE-Healthcare	17-1150-01
LabDiet	Orientbio	5053