Name: a pilot study on the repetitive transcranial magnetic stimulation of aβ and tau levels in rhesus monkey cerebrospinal fluid
Uploaded: 2021-09-03T15:00:00.000+00:00
Duration: 6 min 22 s
Description: Watch this Scientific Journal Video about A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of AÎ² and Tau Levels in Rhesus Monkey Cerebrospinal Fluid at JoVE.com

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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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 and pTau proteins are released into the extracellular space during the neurodegenerative process, resulting in increased tau concentrations in CSF20,29. Therefore, CSF A&#946;1-42, A&#946;42/A&#946;40, tTau, and pTau are confirmed and combined CSF biomarkers in the revised diagnostic criteria of AD1,29.
This study demonstrates that after the rTMS stimulation, the A&#946;42 levels in CSF showed an upward trend at all frequencies. High-frequency rTMS (20 Hz and 40 Hz) increased the A&#946;42 levels to a greater extent than the low frequency. According to previous research30,31, a low level of A&#946;42 in CSF is associated with AD-specific neurodegeneration (i.e., hippocampal atrophy). However, the increase in A&#946; after rTMS stimulation reverses the pathological features of AD, indicating that rTMS may normalize A&#946; levels. A preclinical study indicates that the A&#946; level is regulated by neuronal activity32. Therefore, high-frequency rTMS, vs. low-frequency rTMS, may increase the production of all A&#946; substances, including A&#946;42, by activating neural network activity. In addition, the study found that after 24 h of rTMS at three different frequencies (1 Hz, 20 Hz, and 40 Hz), the pTau level was below the baseline. This indicated a decrease in the abnormal pTau protein, reducing its binding to microtubules and maintaining the normal structure of neurons. However, after high-frequency rTMS, the tTau level of CSF immediately increased and gradually decreased over 24 h. The mechanism underlying this phenomenon is still unclear.
This study objectively confirms the effect of rTMS on A&#946; and tau metabolism in CSF. Compared with other evaluation methods, CSF biomarkers can reflect the metabolism and pathology of the brain, providing a window for the brain. This method is safe and well-tolerated and has great clinical applicability33,34. The most common technique to collect CSF is to perform a lumbar puncture. However, it is challenging to collect CSF several times in a short period, as there are risks of CNS infection and CSF leakage due to the repeated dural puncture35,36.
This protocol uses a novel CSF sampling method, allowing for repeated CSF sampling under fully awake conditions, with low risks of the aforementioned adverse events. The sampling port is placed under the skin so that the monkey cannot scratch the port. Therefore, the CSF can be directly collected through the sampling port rather than by lumbar puncture. The method is convenient and quick and avoids the impact of anesthetics16. Therefore, researchers who need multiple samples of monkey CSF can consider this serial cisterna magna CSF sampling method. To avoid interrupting the process of rTMS, monkey chair adaptive training and rTMS adaptive training are important before beginning the experiment.
Nevertheless, the monkey&#39;s head still has a small range of movement during the experiment even after the training. Hence, it is advisable to use a robot-assisted tracking system, to localize the stimulation sites and position the TMS coil simultaneously when the head moves. This study has some limitations: the animal used here was a normal monkey rather than a pathological model (such as aged canines37), and the sample size was small. However, this pilot study has shown interesting dynamic changes in the levels of A&#946; and tau after rTMS, indicating the potential benefits of rTMS on AD and warranting further investigation.

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

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 A&#946; 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).
A&#946;42 and A&#946;42/A&#946;40 
As shown in Figure 4A, after 1 Hz rTMS stimulation, the A&#946;42 levels gradually increased over 24 h (p &#60; 0.05) and returned to baseline after the washout period. Similarly, after stimulating the bilateral DLPFC of the monkey with rTMS at 20 Hz, the A&#946;42 levels increased with time and reached a peak at 6 h after stimulation (p &#60; 0.05). However, after stimulation with 40 Hz rTMS, the A&#946;42 levels significantly increased immediately at the timepoint of post-rTMS (p &#60; 0.05) and decreased slowly. In general, the high frequencies of rTMS (20 Hz and 40 Hz) increased A&#946;42 levels to a greater extent than the low frequency (1 Hz) (p &#60; 0.05). Moreover, the A&#946;42 levels increased more quickly at the high frequencies, especially at 40 Hz, reached a peak just after stimulation. Moreover, the A&#946;42 level at 40 Hz rose significantly compared with that at 20 Hz (p &#60; 0.05). The ratio of A&#946;42/A&#946;40 showed an upward trend after stimulation with 1 Hz and 20 Hz rTMS and significantly increased from 2 h after rTMS stimulation. Further, it increased to a greater extent after 20 Hz rTMS than with 1 Hz (p &#60; 0.05) (Figure 4B). However, there was no significant difference in the A&#946;42/A&#946;40 ratio at 40 Hz.
pTau and tTau 
Overall, the tTau levels in monkey CSF immediately increased after both 20 Hz and 40 Hz rTMS stimulation (p &#60; 0.05) and decreased gradually (Figure 4C). However, there was no significant difference after 1 Hz rTMS. The pTau level increased immediately and dramatically after the stimulation with 40 Hz rTMS (p &#60; 0.05) and decreased to below baseline level after 24 h (Figure 4D). Additionally, the pTau level showed a downward trend after 1 Hz and 20 Hz rTMS stimulation. Therefore, compared to the other two frequencies (1 Hz and 20 Hz), 40 Hz rTMS showed more significant effects on Tau levels (p &#60; 0.05).
Baseline after washout 
After a 24 h washout period, no significant difference from baseline (p &#62; 0.05) was observed in any CSF biomarker levels.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63005/63005fig01.jpg" /> 
Figure 1: The flow chart for this pilot study. Abbreviation: rTMS = repetitive transcranial magnetic stimulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63005/63005fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63005/63005fig02.jpg" /> 
Figure 2: Minimally invasive catheterization for serial sampling of CSF from cisterna magna. A routine lumbar puncture was followed by a minimally invasive catheterization, in which an epidural catheter penetrated the subarachnoid space and was kept floating in the cisterna magna under the guidance of X-ray (red arrow). A sampling port was left subcutaneously beside the puncture point to allow sampling of the cisterna magna CSF under in a fully conscious animal. Abbreviation: CSF = cerebrospinal fluid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63005/63005fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63005/63005fig03.jpg" /> 
Figure 3: Monkey chair adaptability training. (A) Front; (B) lateral; (C) rTMS adaptive training/sham stimulation. Abbreviation: rTMS = repetitive transcranial magnetic stimulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63005/63005fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63005/63005fig04.jpg" /> 
Figure 4: Effects of rTMS on A&#946; and tau levels in rhesus monkey CSF. The five bars for each frequency represent five timepoints: pre-rTMS, 0 h post-rTMS, 2 h post-rTMS, 6 h post-rTMS, and 24 h post-rTMS. (A) Changes in A&#946;42 level in monkey CSF after rTMS; (B) changes in A&#946;42/A&#946;40 ratio in monkey CSF after rTMS; (C) changes in tTau levels in monkey CSF after rTMS stimulation; (D) Changes in pTau levels in monkey CSF after rTMS. * represents a significant difference from the pre-rTMS level, p &#60; 0.05. # and &#9650; represent significant differences from the level of 1 Hz or 20 Hz at the same timepoint, respectively. p &#60; 0.05, ** p &#60; 0.01, *** p &#60; 0.001, **** represents p &#60; 0.0001. Abbreviations: rTMS = repetitive transcranial magnetic stimulation; CSF = cerebrospinal fluid; tTau = total Tau; pTau = phosphorylated Tau. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63005/63005fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of AÎ² and Tau Levels in Rhesus Monkey Cerebrospinal Fluid at JoVE.com

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

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 ...

a-pilot-study-on-repetitive-transcranial-magnetic-stimulation-tau

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.6K Views.  Sichuan University. Dit protocol maakt gebruik van seriële cisterna magna CSF bemonsteringsmethode om de effecten van rTMS op alfa-bèta- en tau-niveaus bij resusapen te onderzoeken. Deze techniek maakt gebruik van een nieuwe csf-bemonsteringsmethode die herhaalde csf-bemonstering mogelijk maakt onder volledig wakkere omstandigheden met een laag risico op bijwerkingen. De procedure wordt gedemonstreerd door Ying-Qian Zhang, een onderzoeksassistent van de afdeling Fysiologie aan de Southwest Medical University, ...

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

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

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.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Medicine

Neuronavigation-guided Repetitive Transcranial Magnetic Stimulation for Aphasia

Repetitive transcranial magnetic stimulation (rTMS) is widely used for several neurological conditions, as it has gained acknowledgement for its potential therapeutic effects. Brain excitability is non-invasively modulated by rTMS, and rTMS to the language areas has proved its potential effects on treatment of aphasia. In our protocol, we aim to artificially induce virtual aphasia in healthy subjects by inhibiting Brodmann area 44 and 45 using neuronavigational TMS (nTMS), and F3 of the International 10-20 EEG system for conventional TMS (cTMS). To measure the degree of aphasia, changes in reaction time to a picture naming task pre- and post-stimulation are measured and compare the delay in reaction time between nTMS and cTMS. Accuracy of the two TMS stimulation methods is compared by averaging the Talairach coordinates of the target and the actual stimulation. Consistency of stimulation is demonstrated by the error range from the target. The purpose of this study is to demonstrate use of nTMS and to describe the benefits and limitations of the nTMS compared to those of cTMS.

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.

Repetitive transcranial magnetic stimulation (rTMS) is widely used for several neurological conditions, as it has gained acknowledgement for its potential ...

Behavior

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

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&#176;. An in-house water cooling system was used for rTMS for more than 20 min.

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, ...

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Biology

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

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this experiment, we demonstrate the administration of low-frequency repetitive TMS (rTMS) to an optimal stimulation site in the right hemisphere in patients with chronic non-fluent aphasia. A battery of standardized language measures is administered in order to assess baseline performance. Patients are subsequently randomized to either receive real rTMS or initial sham stimulation. Patients in the real stimulation undergo a site-finding phase, comprised of a series of six rTMS sessions administered over five days; stimulation is delivered to a different site in the right frontal lobe during each of these sessions. Each site-finding session consists of 600 pulses of 1 Hz rTMS, preceded and followed by a picture-naming task. By comparing the degree of transient change in naming ability elicited by stimulation of candidate sites, we are able to locate the area of optimal response for each individual patient. We then administer rTMS to this site during the treatment phase. During treatment, patients undergo a total of ten days of stimulation over the span of two weeks; each session is comprised of 20 min of 1 Hz rTMS delivered at 90% resting motor threshold. Stimulation is paired with an fMRI-naming task on the first and last days of treatment. After the treatment phase is complete, the language battery obtained at baseline is repeated two and six months following stimulation in order to identify rTMS-induced changes in performance. The fMRI-naming task is also repeated two and six months following treatment. Patients who are randomized to the sham arm of the study undergo sham site-finding, sham treatment, fMRI-naming studies, and repeat language testing two months after completing sham treatment. Sham patients then cross over into the real stimulation arm, completing real site-finding, real treatment, fMRI, and two- and six-month post-stimulation language testing.

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.

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this ...

Individualized rTMS Treatment for Depression using an fMRI-Based Targeting Method

To achieve greater clinical efficacy, a revolution in treatment for major depressive disorder (MDD) is highly anticipated. Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive and safe neuromodulation technique that immediately changes brain activity. Despite its wide application in the treatment for MDD, the treatment response remains different among individuals, which may be attributable to the inaccurate positioning of the stimulation target. Our study aims to examine whether the functional magnetic resonance imaging (fMRI)-assisted positioning improves the efficacy of rTMS in treating depression. We intend to identify and stimulate the subregion of dorsolateral prefrontal cortex (DLPFC) in MDD with strongest anti-correlation with the subgenual anterior cingulate cortex (sgACC), and to conduct a comparative investigation of this novel method and the traditional 5-cm rule. To achieve more precise stimulation, both methods were applied under the guidance of neuronavigation system. We expected that the TMS treatment with individualized positioning based on resting state functional connectivity may show better clinical efficacy than the 5-cm method.

The present protocol describes the application of repetitive transcranial magnetic stimulation (rTMS), where a subregion of the dorsolateral prefrontal cortex (DLPFC) with the strongest functional anticorrelation with the subgenual anterior cingulate cortex (sgACC) was located as the stimulation target under the assistance of a fMRI-based neuronavigation system.

To achieve greater clinical efficacy, a revolution in treatment for major depressive disorder (MDD) is highly anticipated. Repetitive transcranial ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; 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 A&#946; 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 A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-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 A&#946; 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 A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

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

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial prefrontal cortex (dmPFC) in patients with major depressive disorder (MDD). Technicians used a neuronavigation system to process patient MRIs to generate a 3-dimensional head model. The head model was subsequently used to identify patient-specific stimulatory targets. The dmPFC was stimulated daily for 20 sessions. Stimulation intensity was titrated to address scalp pain associated with rTMS. Weekly assessments were conducted on the patients using the Hamilton Rating Scale for Depression (HamD17) and Beck Depression Index II (BDI-II). Treatment-resistant MDD patients achieved significant improvements on both HAMD and BDI-II. Of note, angled, double-cone coil rTMS at 120% resting motor threshold allows for optimal stimulation of deeper midline prefrontal regions, which results in a possible therapeutic application for MDD. One major limitation of the rTMS field is the heterogeneity of treatment parameters across studies, including duty cycle, number of pulses per session and intensity. Further work should be done to clarify the effect of stimulation parameters on outcome. Future dmPFC-rTMS work should include sham-controlled studies to confirm its clinical efficacy in MDD.

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.

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

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

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