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

Summary

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 Aβ and tau metabolism in rhesus monkey cerebrospinal fluid.

Abstract

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect Aβ 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 Aβ 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 (Aβ₄₂, Aβ₄₂/Aβ₄₀, tTau, pTau). CSF biomarker levels changed with time after stimulation by rTMS. After stimulation, the Aβ₄₂ 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 < 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 < 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 < 0.05). The ratio of Aβ₄₂/Aβ₄₀ showed an upward trend at 1 Hz and 20 Hz (p < 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 Aβ and tau levels in rhesus monkey CSF, while low-frequency (1 Hz) rTMS can only affect Aβ levels.

Introduction

Amyloid-β (Aβ) and tau are important CSF biomarkers. Aβ consists of 42 amino acids (Aβ_1-42), which is the product of transmembrane amyloid precursor protein (APP) hydrolyzed by β- and γ-secretases¹. Aβ_1-42 may aggregate into extracellular amyloid plaques in the brain because of its solubility characteristics^1,2. 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's disease (AD) brain: extracellular amyloid plaques, intracellular NFT formation, and neuron loss⁴. Abnormal concentrations of Aβ and tau present in the early stage of AD, thus allowing early AD diagnosis^5,6.

In 2016, Tsai et al. found that non-invasive light-flickering (40 Hz) reduced the levels of Aβ_1-40 and Aβ_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 Aβ 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 Aβ and improved the overall viability of monoclonal hippocampal HT22 cells in mice¹⁰. After 1 Hz rTMS stimulation, the β-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 reversed^11,12. Bai et al. investigated the effect of rTMS on the Aβ-induced gamma oscillation dysfunction during a working memory test. They concluded that rTMS could reverse Aβ-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 Aβ 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 Aβ and tau levels in rhesus monkey CSF.

Protocol

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'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

1. Use an 8-shaped magnetic field stimulator coil to perform the rTMS stimulation.

2. Animal

1. 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 ± 2 °C, and a 12:12 h light: dark cycle^14,15. Perform all the experiments according to the Guidance for the Care and Use of Laboratory Animals.

3. A serial cisterna magna CSF sampling method

1. Have two trained experimenters perform a catheterization method to sample CSF from the cisterna magna (Figure 2).
2. Positioning
   1. Anesthetize the monkey by an intramuscular injection of 5 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. Monitor its temperature, pulse, respiration, mucous membrane color, and capillary refill time during this stage.
   2. Administer 2 mg/kg morphine via intramuscular injection every 4 hours. 
   3. Place the monkey on an operating table in the lateral decubitus position. Bend the monkey's neck, hunch the back of the monkey, and bring its knees toward the chest.
3. Puncture
   1. 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 "pop" when it enters the lumbar cistern where the ligamentum flavum is housed.
   2. Push the needle again until there is a second "pop" where it enters the dura mater. Withdraw the stylet from the spinal needle and collect drops of CSF.
4. Catheter insertion
   1. Under fluoroscopic guidance, insert the epidural catheter through the puncture needle into the subarachnoid space until it is buoyant in the cisterna magna.
5. Port implantation
   1. 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.
6. CSF collection
   1. Use the bars of the cage to restrain the monkey and keep its back bent.
   2. 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.

4. Monkey chair adaptive training

1. Fix the monkey on the monkey chair before the experiment to avoid interrupting the process of rTMS intervention (Figure 3A,B).
2. Collect CSF for biomarker analysis in the awake state of the monkey to avoid the influence of anesthetic drugs.
3. 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.

5. rTMS adaptive training/sham stimulation

1. 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.
2. 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).
3. Conduct rTMS adaptive training on a monkey chair twice a day, for 30 min each time for a total of 2 weeks.

6. Treatment protocol

1. 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.
   1. Conduct three different sessions of rTMS with a washout period exceeding 24 h^18,19.
      1. 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 days^20,21.
      2. 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.
      3. 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 days^7,22.

7. CSF biomarkers

1. Analyze four CSF biomarkers: Aβ₄₂, Aβ₄₂/Aβ₄₀, tTau, and pTau.

8. CSF collection and index detection method

1. Use a minimally invasive catheterization method to sample the CSF.
2. 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.
3. Collect CSF at 5 timepoints (4 samples each timepoint at 3 min intervals): pre-rTMS, 0 h/2 h/6 h/24 h post-rTMS^23,24,25. Collect a total of 60 samples for 3 frequencies; number and store them in a -80 °C refrigerator for up to 1 month. After the experiment, subject all samples to liquid chip detection according to the manufacturer's instructions (see the Table of Materials).

9. Statistical analysis

1. Present all data as mean ± standard deviation (SD).
2. Perform the Shapiro-Wilk test to test normality in case of a small sample size. Perform two-way repeated-measures ANOVA and Tukey's multiple comparisons test.
   NOTE: A value (two-tailed) < 0.05 was considered statistically significant.

Results

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 gradu...

Discussion

Aβ_1-42, a well-established biomarker of AD, is a CSF core biomarker related to Aβ 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 Aβ₄₂/Aβ₄₀ ratio is a better diagnostic biomarker of AD than Aβ₄₂ alone because it is a better indicator of the AD-type pathology^27,28. Tau...

Materials

Name	Company	Catalog Number	Comments
Anesthesia Puncture Kit for Single Use	Weigao, Shandong, China
CCY-I magnetic field stimulator	YIRUIDE MEDICAL, Wuhan, China
GraphPad Prism version 7.0	GraphPad Software, Inc., San Diego, CA, USA
Human Amyloid Beta and Tau Magnetic Bead Panel	EMD Millipore Corporation, Billerica, MA 01821 USA		liquid chip detection
MILLIPLEX Analyst 5.1	EMD Millipore Corporation, Billerica, MA 01821 USA
Monkey Chair HH-E-1	Brainsight, Cambridge, MA 02140 USA
Zoletil 50	Virbac, France		zolazepam–tiletamine