Activation of Microglia and Attenuation of Amyloid Beta Protein in

Hippocampus Though Sound Induced 40Hz Gamma Oscillation

Tongyu Chen

Nanjing Foreign Language School, Nanjing, Jiangsu, 210008, China

Keywords: Microglia Cell, Gamma Oscillations, Amyloid Beta Protein, Alzheimer’s Disease.

Abstract: Alzheimer's disease (AD) has been a problem for many aged people but the pathogenesis hasn’t been fully

understood yet. The ways to treat it which are found at present are not quite effective. As the AD causes huge

loss to the society each year, it is crucial to develop a new way using sound wave to treat it. A new way of

clearing the amyloid beta protein may help solve this problem and lead the study of AD to a new road. A type

of immune cell called microglia cell, which was found in central nervous system, has the ability to clean

Amyloid beta protein though phagocytosis. This can be a better way to clear the amyloid beta protein by

activation of microglia. Thus, we build a new method for inducing 40Hz gamma oscillation though clicking

sound re is a new way of activating the microglia cell, which is safer and more convenient. So, using sounds

to solve the problem is considered.

1 INTRODUCTION

Amyloid beta protein has been greatly studied in AD

and scientists have tried to find a way to clear it as it

is found in AD patients’ brain and accumulate more

as the disease getting worse. So, it is reckoned that

clearing the amyloid beta protein would help cure the

AD. To better clear the amyloid beta protein, they for

AD therapy in 2012 and 2021, respectively.

However, the results weren’t so perfect as the

symptom of the AD patients, such as memory loss,

showed no remission (Dennis Thompson, Healthday

Reporter).

In a former study the relationship between gamma

oscillation and microglia cell activation was studied.

40Hz gamma oscillation is proved to have the ability

to activate the microglia cell (Martorell, Paulson, Suk

et al 2019). However, the study included invading or

complicated process of inserting electric rods into the

mice brains and light flicker and we hope to look for

a way which is more safe and convenient. Thus, the

use of sound is considered. Sound treatment is a way

which does not invade the organs. Because the use of

light has been accepted in this type of study, we think

of using another kind of senses for inducing 40Hz

gamma oscillation (Hardy, Higgins 1992).

Microglia cell is a kind of neuroglial cell and it

has the ability to degrade the useless protein or other

structures in the brain tissue. After being stimulated,

the microglia cell can be activated and start to clear

the Amyloid beta protein with higher efficiency. In

this test we will find out how to stimulate it and if it

would work.

The Amyloid beta(Aβ) protein is largely found in

AD patients’ brain，and Amyloid beta hypothesis is

now mainly accepted as pathogenesis of AD. The

amyloid beta protein is closely related to the AD and

clearing the amyloid beta protein might have the

ability to cure the AD. The monoclonal antibody of

Amyloid beta protein, Bapineuzumab and

Donanemab, have been applied in clinical study to

activate microglia for Ab clearance.

Changes in gamma oscillations (20–50 Hz) have

been observed in several neurological disorders.

Furthermore, 40Hz oscillations induced by

ontogenetic method in hippocampus has been well

proved to reduce Amyloid beta though microglia

activation.

In this passage, we aim to stimulate the gamma

oscillation by sound waves with different frequencies

and the influence gamma wave has on the clearing

ability of the microglia cell. Then the microglia cell

will be examined to see if it is activated and the

amyloid beta protein is checked to find out if it had

been cleared. If the microglia cell is stimulated but

the Abeta protein isn’t less,we may need to make

632

Chen, T.

Activation of Microglia and Attenuation of Amyloid Beta Protein in Hippocampus Though Sound Induced 40Hz Gamma Oscillation.

DOI: 10.5220/0011250300003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 632-637

ISBN: 978-989-758-595-1

further assumptions. Also, Abeta is less but microglia

isn’t stimulated can happen as well.

In this situation we will need to find out the

relationship between sounds and Abeta protein’s

relationship apart from microglia cell (Iaccarino,

Singer, Martorell, et al, Healthday Reporter 2019).

2 EXPERIMENT APPROACH

2.1 Experiment Materials and

Equipment

30 5XFAD mice, divided into 5 groups, all female

and 2 months old,with similar physical quality

including weight and length ; Amyloid beta 1–40 or

Amyloid beta 1–42 ELISA(Enzyme-Linked Immuno

sorbent Assays) kit (Invitrogen) , mobile phone with

APP for sound maker (The mobile phone is required

to have the ability to make sounds clear enough)

Figure 1: 40Hz gamma oscillation (Martorell, Paulson, Suk et al 2019).

Activation of Microglia and Attenuation of Amyloid Beta Protein in Hippocampus Though Sound Induced 40Hz Gamma Oscillation

633

Table 1:This is a sample figure for the experiment,showing the condition of the mice.

order number of mice/behavior types sleeping time/h food amount/g times moving/h

frequency of

fighting/times

1(20Hz)

2(40Hz)

3(60Hz)

4(80Hz)

5(no sounds)

Figure 2: Visual cortex (Hardy, Higgins 1992).

Special made cages with sound proofing

materials, making sure that the mice won’t be

disturbed by other sounds. The cage is large enough

for the mice to move and water and food is provided

24 hours. The food and water amounts is calculate in

the experiment.

The mice need a certain time to grow and have the

accumulation of the amyloid beta Protein and two

months is suitable. Also, a group of female mice is

easier to live together as they are less likely to fight

with each other like male mice do. The male mice

movements is also more difficult to observe as they

are more likely to move there and here so we may

ignore the change of mice behavior (Cerebral Cortex,

Nicole Barger, Janet Keiter, Cerebral Cortex 2019).

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

634

2.2 Determination of the Effect of the

Sound

First, we need to find out if the sound stimulation can

be emerged in the mice brain including CA1 area and

dentate gyrus area. Stimulation of sounds are given to

the mice and the rate of firing is detected. the sounds

are made while they are having food, sleeping or

running on the spherical treadmill so that the mice

conditions’ influence is lowered. The test will include

20-100Hz sounds which would be used later in sound

experiment. The test of signal will need the electric

rod which will be put in to the hippocampus but in the

later sections invading ways will not be used. In this

section the effect of mice fighting against each other

is also detected using male mice so that we could

make sure the use of female mice is necessary. If the

mice fighting will cause the change of gamma wave

we would be able to find it out in this part of

experiment.

2.3 Determination of Amyloid Beta

Protein

30 5XFAD mice without any treatment are tested

with Amyloid beta ELISA kit from nvitrogen to

detect the original amount of amyloid beta protein.

The CA1 region is isolated from mice, placed in the

salty water, then CA1 tissue is lysed in PBS firstly to

extract PBS (phosphate buffer saline) soluble

Amyloid beta protein and lysed in guanidine

hydrochloric acid to extract the insoluble Amyloid

beta fraction. The soluble Amyloid beta protein

fraction probably contained monomeric and

oligomeric Amyloid beta protein. The Amyloid beta

protein is then collected and determined by ELISA kit

according to manufacture

Figure 3: The area that we used to test the Amyloid beta

protein (The Down-Expression of ACE and IDE

Exacerbates Exogenous Amyloid-Neurotoxicity in CB2R–

/– Mice).

instruction. The antibody from the ELISA kit is

coated to the disc and the extra solution is washed off.

Then the sample is added to it. The disc is washed and

the process is repeated. At last the enzymes are put in

to let it show the colour which can tell how much is

there the amyloid beta protein.

Spectrometer can be used to test how much

amyloid beta protein is found. The sample from mice

and a sample of distilled water is tested at the same

time to get rid off the effect of water refracting light.

This will help making the result rational in the future

(Julie Mazzolini, Sigrid Le Clerc, Glia Wiley 2019,

Ana Badimon, Hayley J. Strasburger, Pinar Ayata,

Nature. 2020).

2.4 Sound Experiment

The sound experiment lasts for 10 days. The sound is

made by a mobile phone and certain app is

downloaded which can make sounds of certain

frequency. The cage will be covered with sound

proofing materials to prevent extra sound influencing

the experiment. The sound source is placed in the

cage directly so the sound is clear enough. The sound

source is specially protected with shelter as mice may

move or damage it. The sound making process will

be controlled remotely for the movement of sound

source and opening of cages may cause influence on

the mice. The source must be designed to minimized

the effect, so it should be as small as possible.

The cage is also huge enough to decrease the

influence of echo sound. During the sound

experiment, each groups of mice receive a different

frequency of sounds.

Group 1 receives a 20 Hz sound frequency, group

2 receives 40Hz, group 3 receives 60Hz, group 4

receives 80Hz and there’s a group 5 which receives

no sounds. The gamma oscillation of sound-treated

mice is also detected to see which sound wave has the

ability to activate the 40Hz gamma oscillation.

It is expected to see the best results in 40Hz sound

wave ’s experiment. The sound wave tend to cause

the gamma wave which has the same frequency.

The mice are exposed to the clicking sound for 2

hours each day during the xperiment. The living

environment of the mice are as quiet as possible to

prevent the noise influence. The cage will be covered

with sound insulation materials to absorb the extra

sound. The cage is also huge enough to decrease the

influence of echo sound. (Hardy, Higgins 1992,

Iaccarino, Singer, Martorell et al 2019, Healthday

Reporter 2019)

After the sound experiment the amyloid beta

protein concentration is tested again. The results will

Activation of Microglia and Attenuation of Amyloid Beta Protein in Hippocampus Though Sound Induced 40Hz Gamma Oscillation

635

show if the amyloid beta protein concentration

decreased after the experiment.

2.5 Detection of Microglia Activation

The microglia activation in hippocampus is also

detected 5 days after the 10 days’ sound experiment.

The cell diameter and the expression of microglial

engulfment associated genes are tested in this section

and detection method is totally following Hannah F’s

method. The rate of the microglia cell which are

activated can show the stimulation ability of the

gamma wave. High number and rate of microglia cell

which are activated show that the gamma wave is

effected for stimulating the microglia cell. To get

better results, tissue from different part of brains

should be tested. In this way we can find out if the

location of the cell will have an effect on the

stimulation signal.

After the test the rate of microglia cell which are

stimulated is checked. Mean and standard deviation

is calculated through the statistics and the numbers

which are too high or low will not be chosen as a

reliable result according to the results quartile number

(both Q1 and Q3).

For the mice, the sound could be some kind of

noise and cause damage to their body. During the test

the heart rate, blood pressure and body temperature

of the mice is also tested. The effect that the

experiment had on the mice can not be estimated

easily and these test could help us to find out .If the

physical quality of the mice changed too much then

the experiment will be less precised for when don’t

know if the microglia cell is stimulated by the sound

or the change of body situation.

After the experiment, the movement and motion

of the mice is also reserved to see if the mice moving

ability has changed. The sound may cause the

insanity of the mice, which would be dangerous for

human patients.

For the observation of the mice, many parts

needed to be taken care of. The basic observation

include the sleeping time of the mice, the amounts of

food the mice had and the times of specific behavior

such as clearing the fur. For other actions such as the

probability of running, they are calculated in the

event of the mice has obvious changes which may

result from the test.

As the number of mice isn’t too large

(number<30), Student’s t test is also used. However,

because the result might not be a Normal distribution

the result may not be precised. The mice are divided

in to groups and Student’s t test, therefore can test if

the mice stimulated microglia cell rate is different

between different groups. (H0:rate in group 1=rate in

group 2,H1:rate in group 1≠rate in group 2,using two-

sided test and see if H0 is rejected.)

2.6 Detection of Amyloid Beta Protein

Clearage by Microglia Cell

Then the amyloid beta protein is tested its location to

see if the protein is cleared. Detection of microglia

phagocytosis of Amyloid beta protein though co-

localization of microglia and anti-Amyloid beta

antibody is processed. The antibody is connected to

fluorescent protein and we look for the place where

fluorescence appears and that’s the place where the

protein goes.

3 EXPECTED RESULTS AND

ANALYSIS

It’s estimated that with different frequencies of sound

waves, the mice are found with different gamma

waves. The mice receiving 40Hz sounds are

discovered to have 40Hz gamma wave. With this

result, we can suggest that the 40Hz sound can cause

he 40Hz gamma oscillation. The 20Hz sounds and

60Hz sounds has less influence, while the 80Hz

sounds has the least effect to cause 40Hz gamma

oscillation. Generally the relationship between the

sound wave frequency and gamma wave frequency is

estimated to be liner.

The result is expected to see the decrease of

amyloid beta protein and the activation of microglia

cell. The microglia cell will show a result of having

larger diameter and a hange on engulfment associated

genes expression, which shows that it is activated.

The results that the amyloid beta protein was inside

the microglia cell should also be detected. If so, then

it proves that the microglia cell can be stimulated by

the gamma wave caused by the sound. Thus, we find

another way of clearing up the Amyloid beta protein

with microglia cells’ cleaning function.

However, there can also be results with the

microglia cell not activated but the 40Hz gamma

wave is detected. This could be caused by a damage

in the brain caused by AD which breaks the link with

microglia cell and other hippocampus cells. Safety

Attention:

1.do not touch the reagent during the experiment

2.do not eat and drink or smoke or using make-up

during the experiment

3.the reagent should be preserved carefully

4.to get the reagent avoid using metal equipment

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636

5.the reagent should be attached with disposable

dropper

6.the reagent shouldn’t be exposed to the air as it

can deteriorate

7.after the experiment the remains should be

managed with care

The safety attention must be followed in the

experiment because they will influence the accuracy

of the result and the statistics.

4 CONCLUSION

The experiment can push forward the study of

amyloid beta protein and the AD. The AD has been

asking for a better therapy for a long time. The use of

sound is both safe and convenient, as it only needs a

way that requires little equipment and funding. This

experiment gives us a new idea of clearing the

amyloid beta protein.

Although the experiment is quite inaccurate, for n

the experiment the sound wave which is 45Hz hasn’t

be considered and the result is not rational, it does

make an approach to the solution to AD.

In the future, if this method is improved to be

more effective, the new way of curing D may be

simply let the patients into a sound giving room and

let them stay for a few days. The patients do not need

to pass the painful course of the treatment, the side

effect and the family can save time and money.

However, there’s still much to we need to explore in

the future: how the microglia cell was stimulated is

still a question. We might find the answers in the

future.

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