AD Model | Construction and Verification Solutions of Alzheimer's Disease Models

Introduction to AD Background

Alzheimer's disease (AD) is a primary neurodegenerative disease of the brain with unknown etiology and complex pathogenesis, and it is one of the most common types of dementia. Clinically, it is mainly characterized by progressive memory loss, cognitive impairment, behavioral abnormalities, and personality disorders. Its typical pathological changes include neuronal loss, senile plaques (SP) formed by amyloid β-protein (Aβ) deposition, and neurofibrillary tangles (NFT) formed by hyperphosphorylation of tau protein [1]. With the accelerating process of population aging, AD has become a hot and focal issue of concern at home and abroad.

AD Modeling Methods

At present, the preparation ideas of commonly used AD animal models are mainly guided by reflecting the pathological, physiological, and clinical manifestations of AD. Establishing models simulating AD pathological characteristics is an important means for AD research. The establishment methods of AD animal models mainly include aging models, transgenic models, exogenous harmful substance injection models, etc. (Table 1). For example, researchers have established AD models characterized by senile plaques induced by direct intracerebral injection of Aβ. Currently, the main injected Aβ are Aβ1-42, Aβ1-40, and Aβ25-35. The injection sites are mainly the unilateral/bilateral hippocampal CA1 area or lateral ventricle, and the injection time can range from 3 days to several weeks [3-4]. Among them, Aβ1-42 is the main form in AD neuropathological research. The nucleus (seed) formed by it initiates the formation of fibers, leading to the classic amyloidosis process.

Next, we will share the method of inducing AD models with Aβ1-42 [3].

1. Preparation of Aβ1-42

1.       Take out the synthetic Aβ1-42 lyophilized powder stored at -80°C and equilibrate at room temperature for 30 minutes;

2.       In a fume hood, suspend Aβ1-42 in 100% HFIP (1.596g/mL) at a concentration of 1mM, mix thoroughly, and aliquot into equal small portions;

3.       Evaporate HFIP from the aliquoted small portions using a vacuum centrifugal evaporator, and store at -20°C after evaporation;

4.       Before use, resuspend the above small portions of Aβ1-42 stored at -20°C in DMSO at a concentration of 5mM;

5.       When in use, dilute to the experimental concentration with normal saline or artificial cerebrospinal fluid as needed for the experiment.

2. Unilateral Lateral Ventricle Cannulation in Mice

1.       After anesthetizing mice with isoflurane inhalation, fix them on a mouse stereotaxic instrument;

2.       Disinfect the top of the cranial bone with 75% alcohol, shave the hair, and disinfect again;

3.       Cut the scalp to expose the skull, and remove the connective tissue on the surface of the skull with hydrogen peroxide. Disinfect again after the skull is dry;

4.       Use a stereotaxic instrument to point the positioning needle to Bregma as the starting point, move the positioning needle to determine the cannula position (0.46mm anterior to Bregma, 1mm bilateral to the midline of the skull, 2.3mm deep), drill a hole with a skull drill, and randomly place the cannula (cannula diameter 0.48mm, inner core diameter 0.3mm) in the left and right lateral ventricles;

5.       Adhere the gap between the cannula and the skull with bone glue, and fix the cannula on the skull with dental cement after the bone glue is dry;

6.       Transfer the mice to an electric blanket, and put them back into the feeding box after waking up, with single cage and single feeding;

7.       During drug injection, pull out the inner core of the catheter, connect the mouse brain cannula and the microinjection needle with a PE tube of appropriate length, and the injection speed of the microinjection pump is 0.5μL/min.

3. Behavioral Tasks

Before the start of the task, inject normal saline or Aβ1-42 into each group of mice that have completed cannula implantation, with the injection speed of the microinjection pump being 0.5μL/min and the injection volume being 2μL. After injection, stop the needle for 5 minutes to prevent backflow of the injection solution, then put back the inner core and lock the nut. Spontaneous alternation Y-maze test and novel location recognition task: 10 minutes after the mice are injected with drugs, start the detection of the spontaneous alternation Y-maze test and the novel location recognition task.

AD Verification Methods

An ideal model should have at least the following three characteristics:

1.       Have the main neuropathological characteristics of AD: SP and NFT.

2.       Exhibit important pathological changes of AD: neuronal death, neural synaptic loss, astrocyte proliferation, etc.

3.       Show cognitive impairment.

In basic AD research and drug discovery, mouse models are important resources for revealing biological mechanisms, verifying molecular targets, and screening potential compounds. Both transgenic and non-transgenic mouse models can obtain different types of AD-like pathology in vivo. Although there are a large number of genetic and biochemical studies on AD pathogenic pathways, as a disease mainly characterized by cognitive impairment, the final readout of any intervention should be the detection of learning and memory [4].

Commonly used test experiments for behavioral analysis of AD mouse models include: contextual fear conditioning, radial arm water maze, and Morris water maze (Figures 2 & 3 & 4). Below, we will take reference [4] as an example to detail the equipment and operation steps of the contextual fear conditioning experiment.

Mice are tested individually in a conditioning chamber (Figure 2), which is located in a sound-attenuating box with a transparent plexiglass window for video recording of the experiment. The conditioning chamber has a 36-bar shock grid floor, which is removable. After each subject, it is cleaned with 75% ethanol and then with water. To provide background white noise (72dB), a single computer fan is installed on one side of the sound-attenuating box.

Before the behavioral experiment, handle the mice once a day for 3 consecutive days. Only one animal is placed in the test room at a time. Place it in the conditioning chamber for 2 minutes, then emit a discrete tone (CS) at 2800Hz and 85dB for 30 seconds. During the last 2 seconds of the tone, the mouse receives a foot shock (US, 0.50-0.80mA, duration 2 seconds) through the floor strips, and the intensity of the foot shock can be adjusted to induce stronger memory. After the CS/US pairing, the mouse is placed in the conditioning chamber for another 30 seconds and then returned to the cage. Freezing behavior can be scored using dedicated software. Based on experience, it is useful to double-check the freezing time through manual scoring. Twenty-four hours after training, the mice are returned to the conditioning chamber. The freezing time is 5 consecutive minutes. Thirty-six hours after training, the mice are transferred to a new conditioning box. A new environment is created by inserting a triangular cage with a smooth floor and a vanilla scent. After 2 minutes (pre-CS test), the mice are exposed to a tone with the same characteristics as that used in training for 3 minutes (CS test), and the freezing time is measured. Usually, 15-20 animals are used in each condition.

An important control to be performed during the test of fear memory is the sensory threshold assessment. Changes in sensory perception of electric shock may be due to experimental manipulation leading to misattribution of the observed phenotypic memory. Deliver a series of single foot shocks to animals placed on the same grid used for fear conditioning. Initially, a 0.1mA shock is applied for 1 second to assess the animal's withdrawal, jumping, and vocalization behaviors. Every 30 seconds, the shock intensity is increased by 0.1-0.7mA, then returned to 0mA in 0.1mA increments every 30 seconds. The vocalization, withdrawal, and jumping thresholds of each animal are quantified by averaging the shock intensities at which each animal exhibits behavioral responses to the foot shock.

AD Treatment

It is reported that there are about 24 million Alzheimer's disease cases worldwide. By 2050, the total number of dementia patients is estimated to increase fourfold. Although AD is a public health problem, so far, only two classes of drugs have been approved for the treatment of AD, including cholinesterase inhibitors (naturally derived, synthetic, and hybrid analogs) and N-methyl-D-aspartate (NMDA) antagonists [5].

Several physiological processes in AD damage acetylcholine-producing cells and reduce cholinergic transmission through the brain. Cholinesterase inhibitors (AChEIs) are divided into reversible, irreversible, and pseudo-reversible types. They work by blocking cholinesterase (AChE) and butyrylcholinesterase (BChE) from decomposing ACh, leading to an increase in ACh levels in the synaptic cleft. On the other hand, excessive activation of NMDAR leads to an increase in the level of intracellular calcium influx, thereby promoting cell death and synaptic dysfunction. NMDAR antagonists prevent excessive activation of NMDAR glutamate receptors, thereby blocking calcium influx and restoring their normal activity. Although these two classes of drugs are effective, they only treat the symptoms of Alzheimer's disease but cannot cure or prevent the disease.

Establishing an ideal animal model to simulate human disease status is conducive to in-depth research on the etiology, pathogenesis, prevention, and treatment of AD. At present, due to the inability to establish a perfect AD animal model, when conducting drug screening, using two or more AD animal models simultaneously is more convincing than using a single model, and can further confirm the effectiveness of the drug.

References

[1] Puzzo D, Gulisano W, Palmeri A, et al. Rodent models for Alzheimer's disease drug discovery[J]. Expert opinion on drug discovery, 2015, 10(7):703-711.

[2] Breijyeh Z, Karaman R. Comprehensive Review on Alzheimer's Disease: Causes and Treatment [J]. Molecules, 2020, 25, (24).

[3] Lü J Y. The mechanism of Aβ1-42 or Aβ1-15 improving memory in APP/PS1/Tau transgenic Alzheimer's disease mice[D]. East China Normal University, 2023.

[4] A D P, B L L, A A P, et al. Behavioral assays with mouse models of Alzheimer's disease: Practical considerations and guidelines-ScienceDirect[J]. Biochemical Pharmacology, 2014, 88(4):450-467.

[5] Breijyeh Z, Karaman R. Comprehensive Review on Alzheimer's Disease: Causes and Treatment[J]. Molecules, 2020, 25:5789.

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