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 (Aβ1-42), which is the product of transmembrane amyloid precursor protein (APP) hydrolyzed by β- and γ-secretases1. Aβ1-42 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'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 diagnosis5,6.
In 2016, Tsai et al. found that non-invasive light-flickering (40 Hz) reduced the levels of Aβ1-40 and Aβ1-42 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 reversed11,12. 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
- Use an 8-shaped magnetic field stimulator coil to perform the rTMS stimulation.
2. Animal
- 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 cycle14,15. Perform all the experiments according to the Guidance for the Care and Use of Laboratory Animals.
3. A serial cisterna magna CSF sampling method
- Have two trained experimenters perform a catheterization method to sample CSF from the cisterna magna (Figure 2).
- Positioning
- 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.
- Administer 2 mg/kg morphine via intramuscular injection every 4 hours.
- 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.
- Puncture
- 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.
- 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.
- Catheter insertion
- Under fluoroscopic guidance, insert the epidural catheter through the puncture needle into the subarachnoid space until it is buoyant in the cisterna magna.
- Port implantation
- 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.
- 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.
- CSF collection
- Use the bars of the cage to restrain the monkey and keep its back bent.
- 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
- Fix the monkey on the monkey chair before the experiment to avoid interrupting the process of rTMS intervention (Figure 3A,B).
- Collect CSF for biomarker analysis in the awake state of the monkey to avoid the influence of anesthetic drugs.
- 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
- 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.
- 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).
- 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
- 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.
- Conduct three different sessions of rTMS with a washout period exceeding 24 h18,19.
- 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.
- 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.
- 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.
- Conduct three different sessions of rTMS with a washout period exceeding 24 h18,19.
7. CSF biomarkers
- Analyze four CSF biomarkers: Aβ42, Aβ42/Aβ40, tTau, and pTau.
8. CSF collection and index detection method
- Use a minimally invasive catheterization method to sample the CSF.
- 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.
- 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 °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
- Present all data as mean ± standard deviation (SD).
- 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.
Representative 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 gradually increased over 24 h (p < 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 Aβ42 levels increased with time and reached a peak at 6 h after stimulation (p < 0.05). However, after stimulation with 40 Hz rTMS, the Aβ42 levels significantly increased immediately at the timepoint of post-rTMS (p < 0.05) and decreased slowly. In general, the high frequencies of rTMS (20 Hz and 40 Hz) increased Aβ42 levels to a greater extent than the low frequency (1 Hz) (p < 0.05). Moreover, the Aβ42 levels increased more quickly at the high frequencies, especially at 40 Hz, reached a peak just after stimulation. Moreover, the Aβ42 level at 40 Hz rose significantly compared with that at 20 Hz (p < 0.05). The ratio of Aβ42/Aβ40 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 < 0.05) (Figure 4B). However, there was no significant difference in the Aβ42/Aβ40 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 < 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 < 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 < 0.05).
Baseline after washout
After a 24 h washout period, no significant difference from baseline (p > 0.05) was observed in any CSF biomarker levels.
Figure 1: The flow chart for this pilot study. Abbreviation: rTMS = repetitive transcranial magnetic stimulation. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 3: Monkey chair adaptability training. (A) Front; (B) lateral; (C) rTMS adaptive training/sham stimulation. Abbreviation: rTMS = repetitive transcranial magnetic stimulation. Please click here to view a larger version of this figure.
Figure 4: Effects of rTMS on Aβ 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 Aβ42 level in monkey CSF after rTMS; (B) changes in Aβ42/Aβ40 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 < 0.05. # and ▲ represent significant differences from the level of 1 Hz or 20 Hz at the same timepoint, respectively. p < 0.05, ** p < 0.01, *** p < 0.001, **** represents p < 0.0001. Abbreviations: rTMS = repetitive transcranial magnetic stimulation; CSF = cerebrospinal fluid; tTau = total Tau; pTau = phosphorylated Tau. Please click here to view a larger version of this figure.
Discussion
Aβ1-42, 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 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 Aβ1-42, Aβ42/Aβ40, 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 Aβ42 levels in CSF showed an upward trend at all frequencies. High-frequency rTMS (20 Hz and 40 Hz) increased the Aβ42 levels to a greater extent than the low frequency. According to previous research30,31, a low level of Aβ42 in CSF is associated with AD-specific neurodegeneration (i.e., hippocampal atrophy). However, the increase in Aβ after rTMS stimulation reverses the pathological features of AD, indicating that rTMS may normalize Aβ levels. A preclinical study indicates that the Aβ level is regulated by neuronal activity32. Therefore, high-frequency rTMS, vs. low-frequency rTMS, may increase the production of all Aβ substances, including Aβ42, 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 Aβ 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'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 Aβ and tau after rTMS, indicating the potential benefits of rTMS on AD and warranting further investigation.
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
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