This protocol uses serial cisterna magna CSF sampling method to explore the effects of rTMS on alpha-beta and tau levels in rhesus monkey. This technique uses a novel CSF sampling method allowing for repeated CSF sampling under fully awake conditions with low risks of adverse events. Demonstrating the procedure will be Ying-Qian Zhang, a research assistant from the Department of Physiology at Southwest Medical University, and Hui-Xin Tan, a graduate student from Sichuan University.
After administering analgesia and confirming successful anesthetization, place the five-year-old male rhesus monkey on the operating table in the lateral decubitus position and disinfect the area around the lower back. Hunch its back and bring its knees toward the chest. Insert a spinal needle between the lumbar vertebrae L4 to L5 and push it in until there is a pop indicating entry into the lumbar cistern where the ligamentum flavum is housed.
Continue to push the needle further until there is a second pop indicating entry into the dura mater, then withdraw the stylet from the spinal needle and collect drops of CSF. Next, perform catheter insertion under fluoroscopic guidance by inserting the epidural catheter through the puncture needle into the subarachnoid space until it is buoyant in the cisterna magna. Then for port implantation, make a five centimeter incision from the puncture site toward the head and isolate the skin from the subcutaneous tissue to place the sampling port.
Connect the port to the end of the epidural catheter, implant the port under the skin and suture the incision. For CSF collection, use the cage bars to restrain the monkey and keep its back bent. Then wipe with an alcohol swab and insert the syringe into the center of the sampling port to extract CSF from the cisterna magna through the catheter.
Discard the first 200 microliters of CSF, then collect one milliliter of CSF for analysis. Fix the monkey on the monkey chair before the experiment to avoid interruption during the rTMS intervention, then collect CSF for biomarker analysis when the monkey is awake to avoid the influence of anesthetic drugs. On the third day after the subarachnoid catheterization and two weeks before the start of the experiment, subject the monkey to adaptive training with the monkey chair twice a day for 30 minutes each time.
Conduct the rTMS adaptive training or sham stimulation one week after the adaptive training with the monkey chair and one week before the start of the formal experiment to avoid hindering the progress of the experiment by vibrations and sounds during the stimulation process. For sham stimulation, use a sham coil that produces vibration and sound but does not generate a magnetic field. After stimulation, offer food to the monkey to help it adapt to the process.
Localize the bilateral dorsolateral prefrontal cortex according to the international 10 to 20 system and conduct three different sessions of rTMS using three different frequencies without a washout period exceeding 24 hours. Perform the first, second, and third sessions twice a day for three consecutive days. To analyze the four CSF biomarkers, use a minimally invasive catheterization method to collect the CSF.
Collect CSF at five time points with four samples at each time point at three-minute intervals. After collecting a total of 60 samples for three frequencies, number and store them in a minus 80 degree Celsius refrigerator for up to one month. After the experiment, subject all samples to liquid chip detection according to the manufacturer's instructions.
After one hertz rTMS stimulation, Abeta42 levels gradually increased over 24 hours. Similarly with rTMS at 20 hertz, the Abeta42 levels increased with time and reached a peak at six hours after stimulation. In contrast, after 40 hertz stimulation, Abeta42 levels significantly increased immediately at the post-rTMS time point, then decreased slowly.
The ratio of Abeta42 to Abeta40 showed an upward trend after stimulation with one and 20 hertz rTMS and significantly increased two hours after stimulation. However, there was no significant difference in the ratio at 40 hertz. The tTau levels in CSF immediately increased after 20 and 40 hertz rTMS stimulation and decreased gradually.
However, there was no significant difference after one hertz stimulation. The pTau level immediately increased after 40 hertz stimulation and decreased to below baseline level after 24 hours. In contrast, for the one and 20 hertz stimulation, the pTau level showed a downward trend.
Researchers requiring multiple samples of monkey CSF can consider this serial cisterna magna CSF sampling method.
