Name: A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of Aβ and Tau Levels in Rhesus Monkey Cerebrospinal Fluid (Video) | JoVE Journal
Uploaded: 2021-09-03T15:00:00.000+00:00
Duration: 6 min 22 s
Description: Explore repetitive transcranial magnetic stimulation effects on amyloid-beta and tau levels in rhesus monkey cerebrospinal fluid using a novel awake sampling method.

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5 minutes

The authors would like to thank Sichuan Green-House Biotech Co., Ltd for providing the monkey chair and other relative devices. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

All the experiments were performed under the Guidance for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People&#39;s Republic of China, as well as the principles of the Basel Declaration. Approval was given by the Animal Care Committee of the Sichuan University West China Hospital (Chengdu, China). Figure 1 shows the single-blind, self-controlled study design used here.
1. rTMS devices
<ol>
	<li>Use an 8-shaped magnetic field stimulator coil to perform the rTMS stimulation.</li>
</ol>
2. Animal
<ol>
	<li>Keep the male rhesus monkey (Macaca mulatta, 5 kg, 5 years old) in an individual home cage with free access to tap water and standard chow. Ensure that environmental conditions are controlled to provide a relative humidity of 60-70%, a temperature of 24 &#177; 2 &#176;C, and a 12:12 h light: dark cycle14,15. Perform all the experiments according to the Guidance for the Care and Use of Laboratory Animals.</li>
</ol>
3. A serial cisterna magna CSF sampling method
<ol>
	<li>Have two trained experimenters perform a catheterization method to sample CSF from the cisterna magna (Figure 2).</li>
	<li>Positioning
	<ol>
		<li>Anesthetize the monkey by an intramuscular injection of 5&#160;mg/kg zolazepam-tiletamine (see the Table of Materials). To ensure successful anesthetization of the monkey, look for deep and slow breathing, dull or absent cornea reflex, and relaxation of the muscles of the extremities.&#160;Monitor its temperature, pulse, respiration, mucous membrane color, and capillary refill time during this stage.</li>
		<li>Administer 2 mg/kg morphine via intramuscular injection every 4 hours.&#160;</li>
		<li>Place the monkey on an operating table in the lateral decubitus position. Bend the monkey&#39;s neck, hunch the back of the monkey, and bring its knees toward the chest.</li>
	</ol>
	</li>
	<li>Puncture
	<ol>
		<li>For disinfection, prepare the area around the lower back using aseptic technique. Insert a spinal needle between the lumbar vertebrae L4/L5, push it in until there is a &#34;pop&#34; when it enters the lumbar cistern where the ligamentum flavum is housed.</li>
		<li>Push the needle again until there is a second &#34;pop&#34; where it enters the dura mater. Withdraw the stylet from the spinal needle and collect drops of CSF.</li>
	</ol>
	</li>
	<li>Catheter insertion
	<ol>
		<li>Under fluoroscopic guidance, insert the epidural catheter through the puncture needle into the subarachnoid space until it is buoyant in the cisterna magna.</li>
	</ol>
	</li>
	<li>Port implantation
	<ol>
		<li>Make a 5 cm incision from the puncture site to the direction of the head and isolate the skin from subcutaneous tissue to place the sampling port. Connect the port to the end of the epidural catheter and implant the port under the skin; then, suture the incision. Disinfect the wound daily to prevent infection. 
		NOTE: The monkey fully recovers on the day after surgery.</li>
	</ol>
	</li>
	<li>CSF collection
	<ol>
		<li>Use the bars of the cage to restrain the monkey and keep its back bent.</li>
		<li>Insert a syringe into the center of the sampling port to extract the CSF from the cisterna magna through the catheter. Discard the first 0.2 mL of CSF (the total volume of the catheter and port is 0.1 mL), and then collect 1 mL of CSF for analysis16.</li>
	</ol>
	</li>
</ol>
4. Monkey chair adaptive training
<ol>
	<li>Fix the monkey on the monkey chair before the experiment to avoid interrupting the process of rTMS intervention (Figure 3A,B).</li>
	<li>Collect CSF for biomarker analysis in the awake state of the monkey to avoid the influence of anesthetic drugs.</li>
	<li>On the third day after the subarachnoid catheterization, 2 weeks before the start of the experiment, subject the monkey to adaptive training with the monkey chair, twice a day, for 30 min each time.</li>
</ol>
5. rTMS adaptive training/sham stimulation
<ol>
	<li>Conduct the rTMS adaptive training/sham stimulation one week after the adaptive training with the monkey chair, one week before the start of the formal experiment to avoid hindering the progress of the experiment because of vibrations and sounds during the stimulation process.</li>
	<li>Use a sham coil (which only produces vibration and sound and does not generate a magnetic field) to stimulate the monkey. Offer food to the monkey after stimulation to help it adapt to the process (Figure 3C).</li>
	<li>Conduct rTMS adaptive training on a monkey chair twice a day, for 30 min each time for a total of 2 weeks.</li>
</ol>
6. Treatment protocol
<ol>
	<li>Use three different frequencies (1 Hz/20 Hz/40 Hz) of rTMS to stimulate the bilateral-DLPFC (R-L-DLPFC) of the monkey, as described previously17. Localize the DLPFC according to the international 10-20 system.
	<ol>
		<li>Conduct three different sessions of rTMS with a washout period exceeding 24 h18,19.
		<ol>
			<li>For the first period, use the following parameters: a frequency of 1 Hz for rTMS, a pattern of rTMS composed of 20 burst trains, 20 pulses with 10 s inter-train intervals between trains, and an intensity of stimulation of 100% of the average resting motor threshold (RMT), twice a day for three consecutive days20,21.</li>
			<li>For the second period, use the following parameters: trains of high frequency (20 Hz) rTMS with 100% RMT for 2 s duration with 28 s inter-train intervals, a total of 2,000 stimuli (40 stimuli/train, 50 trains) each session, twice a day for three consecutive days22.</li>
			<li>For the third period, use the following parameters: trains of gamma-frequency (40 Hz) rTMS with 100% RMT delivered in 1 s duration separated by 28 s inter-train intervals. Keep the total number of pulses for each session the same as with 20 Hz rTMS, twice a day for three consecutive days7,22.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
7. CSF biomarkers
<ol>
	<li>Analyze four CSF biomarkers: A&#946;42, A&#946;42/A&#946;40, tTau, and pTau.</li>
</ol>
8. CSF collection and index detection method
<ol>
	<li>Use a minimally invasive catheterization method to sample the CSF.</li>
	<li>Use the bars of the cage to restrain the monkey and keep its back bent. Instruct the other operator to insert a syringe into the center of the sampling port, ensuring that CSF is extracted through the catheter.</li>
	<li>Collect CSF at 5 timepoints (4 samples each timepoint at 3 min intervals): pre-rTMS, 0 h/2 h/6 h/24 h post-rTMS23,24,25. Collect a total of 60 samples for 3 frequencies; number and store them in a -80 &#176;C refrigerator for up to 1 month. After the experiment, subject all samples to liquid chip detection according to the manufacturer&#39;s instructions (see the Table of Materials).</li>
</ol>
9. Statistical analysis
<ol>
	<li>Present all data as mean &#177; standard deviation (SD).</li>
	<li>Perform the Shapiro-Wilk test to test normality in case of a small sample size. Perform two-way repeated-measures ANOVA and Tukey&#39;s multiple comparisons test. 
	NOTE: A value (two-tailed) &#60; 0.05 was considered statistically significant.</li>
</ol>

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</ol>

The authors have no conflicts of interest to declare.

A&#946;1-42, a well-established biomarker of AD, is a CSF core biomarker related to A&#946; metabolism and amyloid plaque formation in the brain and has been widely used in clinical trials and the clinic26. Recent studies have shown that the CSF A&#946;42/A&#946;40 ratio is a better diagnostic biomarker of AD than A&#946;42 alone because it is a better indicator of the AD-type pathology27,28. Tau...

Amyloid-&#946; (A&#946;) and tau are important CSF biomarkers. A&#946; consists of 42 amino acids (A&#946;1-42), which is the product of transmembrane amyloid precursor protein (APP) hydrolyzed by &#946;- and &#947;-secretases1. A&#946;1-42 may aggregate into extracellular amyloid plaques in the brain because of its solubility characteristics1,2. Tau is a microtubule-associated protein that is mainly present in axons and is involved in anterograde axonal transport3. Abnormal tau hyperphosphorylation is mainly induced by the imbalance between kinases and phosphatases, resulting in the detachment of tau from microtubules and the formation of neurofibrillary tangles (NFT)1. The concentration of tau increases in the CSF because tau and phosphorylated tau proteins (pTau) are released into the extracellular space during the neurodegenerative process. Previous studies have shown that CSF biomarkers are relevant to the three main pathological changes of the Alzheimer&#39;s disease (AD) brain: extracellular amyloid plaques, intracellular NFT formation, and neuron loss4. Abnormal concentrations of A&#946; and tau present in the early stage of AD, thus allowing early AD diagnosis5,6.
In 2016, Tsai et al. found that non-invasive light-flickering (40 Hz) reduced the levels of A&#946;1-40 and A&#946;1-42 in the visual cortex of pre-depositing mice7. Recently, they further reported that auditory tone stimulation (40 Hz) improved recognition and spatial memory, reduced amyloid protein levels in the hippocampus and auditory cortex (AC) of 5XFAD mice, and decreased pTau concentrations in the P301S tauopathy model8. These results indicate that non-invasive techniques could impact A&#946; and tau metabolism.
As a non-invasive tool, transcranial magnetic stimulation (TMS) could electrically stimulate neural tissue, including the spinal cord, peripheral nerves, and cerebral cortex9. Moreover, it can modify the excitability of the cerebral cortex at the stimulated site and in the functional connections. Therefore, TMS has been used in the treatment of neurodegenerative disorders and prognostic and diagnostic tests. The most common form of clinical intervention in TMS, rTMS, can induce cortex activation, modify the excitability of the cortex, and regulate cognitive/motor function.
It was reported that 20 Hz rTMS had an in vitro neuroprotective effect against oxidative stressors, including glutamate and A&#946; and improved the overall viability of monoclonal hippocampal HT22 cells in mice10. After 1 Hz rTMS stimulation, the &#946;-site APP-cleaving enzyme 1, APP, and its C-terminal fragments in the hippocampus were considerably reduced. Notably, the impairment of long-term potentiation, spatial learning, and memory in hippocampal CA1 was reversed11,12. Bai et al. investigated the effect of rTMS on the A&#946;-induced gamma oscillation dysfunction during a working memory test. They concluded that rTMS could reverse A&#946;-induced dysfunction, resulting in potential benefits for working memory13. However, there are few reports on the effects of rTMS on tau metabolism and the dynamic changes in A&#946; and tau in CSF before and after rTMS. This protocol describes the procedure for investigating the effects of rTMS at different frequencies (low frequency, 1 Hz; high frequencies,20 Hz, and 40 Hz) on A&#946; and tau levels in rhesus monkey CSF.

<table><tbody><tr><td>Anesthesia Puncture Kit for Single Use</td><td>Weigao, Shandong, China</td><td></td><td></td></tr><tr><td>CCY-I magnetic field stimulator</td><td>YIRUIDE MEDICAL, Wuhan, China</td><td></td><td></td></tr><tr><td>GraphPad Prism version 7.0</td><td>GraphPad Software, Inc., San Diego, CA, USA</td><td></td><td></td></tr><tr><td>Human Amyloid Beta and Tau Magnetic Bead Panel</td><td>EMD Millipore Corporation, Billerica, MA 01821 USA</td><td></td><td>liquid chip detection</td></tr><tr><td>MILLIPLEX Analyst 5.1</td><td>EMD Millipore Corporation, Billerica, MA 01821 USA</td><td></td><td></td></tr><tr><td>Monkey Chair HH-E-1</td><td>Brainsight, Cambridge, MA 02140 USA</td><td></td><td></td></tr><tr><td>Zoletil 50</td><td>Virbac, France</td><td></td><td>zolazepam&amp;ndash;tiletamine</td></tr></tbody></table>

a pilot study on the repetitive transcranial magnetic stimulation of a&#946; and tau levels in rhesus monkey cerebrospinal fluid

A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of A&#946; and Tau Levels in Rhesus Monkey Cerebrospinal Fluid

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in the brain. As a non-invasive technique, repetitive transcranial magnetic stimulation (rTMS) has been applied for the treatment of neurodegenerative disorders. This study explored the effects of rTMS on A&#946; and tau levels in rhesus monkey cerebrospinal fluid (CSF). This is a single-blind, self-controlled study. Three different frequencies (low frequency, 1 Hz; high frequencies, 20 Hz and 40 Hz) of rTMS were used to stimulate the bilateral-dorsolateral prefrontal cortex (DLPFC) of the rhesus monkey. A catheterization method was used to collect CSF. All samples were subjected to liquid chip detection to analyze CSF biomarkers (A&#946;42, A&#946;42/A&#946;40, tTau, pTau). CSF biomarker levels changed with time after stimulation by rTMS. After stimulation, the A&#946;42 level in CSF showed an upward trend at all frequencies (1 Hz, 20 Hz, and 40 Hz), with more significant differences for the high-frequencies (p &lt; 0.05) than for the low frequency. 
After high-frequency rTMS, the total Tau (tTau) level of CSF immediately increased at the post-rTMS timepoint (p &lt; 0.05) and gradually decreased by 24 h. Moreover, the results showed that the level of phosphorylated Tau (pTau) increased immediately after 40 Hz rTMS (p &lt; 0.05). The ratio of A&#946;42/A&#946;40 showed an upward trend at 1 Hz and 20 Hz (p &lt; 0.05). There was no significant difference in the tau levels with low-frequency (1 Hz) stimulation. Thus, high-frequencies (20 Hz and 40 Hz) of rTMS may have positive effects on A&#946; and tau levels in rhesus monkey CSF, while low-frequency (1 Hz) rTMS can only affect A&#946; levels.

The results showed that rTMS could affect the Aβ and tau levels in rhesus monkey CSF. CSF biomarker levels changed with time after rTMS stimulation at different frequencies (1 Hz, 20 Hz, and 40 Hz).
Aβ42 and Aβ42/Aβ40 
As shown in Figure 4A, after 1 Hz rTMS stimulation, the Aβ42 levels gradually increased over 24 h (p...

Here, we describe the procedure for a pilot study to explore the effect of repetitive transcranial magnetic stimulation with different frequencies (1 Hz/20 Hz/40 Hz) on A&#946; and tau metabolism in rhesus monkey cerebrospinal fluid.

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in ...

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Research

JoVE Journal

Neuroscience

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system.  Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Source: Taube, W., et.al., Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans. J. Vis. Exp. (2017)This video demonstrates a non-invasive method to assess changes in corticomotoneuronal transmission in humans following repetitive transcranial magnetic stimulation (rTMS). The study introduces an electrophysiological technique that differentiates fast, direct corticospinal pathways from polysynaptic connections.

Repetitive Transcranial Magnetic Stimulation-Induced Changes in Human Corticomotoneuronal Synapses

Source: Beom, J., et al. Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain. J. Vis. Exp. (2016)This video demonstrates the application of repetitive transcranial magnetic stimulation (rTMS) in anesthetized rats to modulate cortical excitability. The procedure involves determining the motor threshold (MT) to establish baseline stimulation, followed by applying rTMS at an intensity above the MT to induce neuronal depolarization. This stimulation triggers...

Modulating Cortical Excitability in a Rat Using Repetitive Transcranial Magnetic Stimulation

Source: Dunlop, K., et al. MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder. J. Vis. Exp. (2015)This video demonstrates the procedure for MRI-guided repetitive transcranial magnetic stimulation (rTMS) targeting the dorsomedial prefrontal cortex (dmPFC) to treat depression.

MRI-Guided Repetitive Transcranial Magnetic Stimulation to the Dorsomedial Prefrontal Cortex

We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia.  After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.

Utilizing Repetitive Transcranial Magnetic Stimulation to Improve Language Function in Stroke Patients with Chronic Non-fluent Aphasia

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder

This study is designed to test the hypothesis that neuronavigational system-guided transcranial magnetic stimulation has higher accuracy for targeting the intended target as demonstrated by eliciting a greater degree of virtual aphasia in healthy subjects, measured by delay in reaction time to picture naming.

Neuronavigation-guided Repetitive Transcranial Magnetic Stimulation for Aphasia

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45°. An in-house water cooling system was used for rTMS for more than 20 min.

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

This protocol presents a more precise and effective method of repetitive transcranial magnetic stimulation of the dorsolateral and dorsomedial prefrontal cortices during an experimental task using neuronavigation and robotic systems. Based on the results, crucial implications are suggested for future online stimulation research.

Online Repetitive Transcranial Magnetic Stimulation of Dorsomedial and Dorsolateral Prefrontal Cortex in Cognition Decision Making, and Cognitive Dissonance

This protocol presents a guide for the precise and patient-centered application of repetitive transcranial magnetic stimulation for treatment-resistant major depression. Details are provided on procedures for accurate coil localization, adjustable stimulus dosing, rigorous safety measures, and strategies that maximize comfort, reproducibility, and long-term clinical effectiveness across acute and maintenance sessions.

Conventional Repetitive Transcranial Magnetic Stimulation for Depression: A Step-by-Step Protocol

Explore repetitive transcranial magnetic stimulation effects on amyloid-beta and tau levels in rhesus monkey cerebrospinal fluid using a novel awake sampling method.

A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of Aβ and Tau Levels in Rhesus Monkey Cerebrospinal Fluid

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