Real-Time Dynamic Collection of Hippocampal Extracellular Fluid from Conscious Rats Using a Microdialysis System

The experimental protocol was conducted in accordance with the requirements of the Use of Laboratory Animals and Institutional Animal Care and Use Committee at Chengdu University of Traditional Chinese Medicine (Record number: 2021-11). Male Sprague Dawley (SD) rats (280 ± 20 g, 6-8 weeks old) were used for the present study.

1. Brain microdialysis probe implantation surgery

1. Use 3% and 1.5% isoflurane for the induction and maintenance of rat anesthesia, respectively, using an animal anesthesia system in an air-oxygen mixture at 0.6 L/min. Make sure that the rats are deeply anesthetized without both a pain reflex and a corneal reflex. Use veterinary ointment on the eyes to prevent dryness while under anesthesia.
2. Remove the fur on the anesthetized rat's skull with an electric shaver in the preparation area. Then, fix the anesthetized rat on a stereotaxic brain locator. Disinfect the surgical site before the operation by applying povidone-iodine and ethanol 3x to the region of surgery with a sterile cotton ball. Apply bupivacaine topically for local analgesia. NOTE: The entire process of microdialysis-based HECF sample acquisition is illustrated in Figure 1.
3. Make a 1.5 cm craniofacial incision down the middle with surgical scissors and remove the periosteum using surgical scissors and ophthalmic forceps.
4. Consider the bregma as the basal position and pierce the endocranium to drill a 2 mm aperture at the anteroposterior (AP) position (-2 mm), the mediolateral (ML) position (-3.5 mm), and the dorsoventral (DV) position (-3.5 mm) (the hippocampal CA1 region) using a cranial drill.
5. Fix the catheter stylet on the gripper of a stereotaxic brain locator and adjust the position of the microdialysis casing at the AP (-2 mm), ML (-3.5 mm), and DV (0 mm) positions. Adjust the DV value of the brain stereo locator, and implant the microdialysis casing into the CA1 region at a depth of 3.5 mm.
   NOTE: Maintain the temperature of the animal at 37 °C during the operation using an animal temperature maintainer.
6. Drill three more apertures with a 2 mm diameter such that the three apertures form a triangle in which the probe aperture is centrally located. Implant screws into the apertures at a depth of 1 mm.
7. Fix the probe catheter with dental cement and use a 4-0 surgical suture to close the skin. See Figure 2 for the placement of the probe.
8. Place the rat in cages for 7 days to recover. Locally infiltrate bupivacaine (1.5 mg/kg) once daily post-surgery. Provide food and water ad libitum. Use sodium hyaluronate eye drops 3x a day to prevent dryness after the operation.
   ​NOTE: Perform all the procedures in a sterile surgical room. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency under a 37 °C condition. Do not return the animal that has undergone surgery to the company of other animals until fully recovered.

2. Microdialysis system connection and probe check

1. Connect the microdialysis pump, microsyringe, awake activity device, and cryogenic sample collector according to the manufacturer's instructions. Install the microsyringe with ACSF on the microdialysis pump and set the microdialysis pump to a rate of 1 µL/min to discharge the air in the pipeline.
2. Connect the pipeline and brain microdialysis probe (membrane: PAES; membrane length: 4 mm; membrane OD: 0.5 mm; cut-off: 20 kDa; shaft length: 14 mm). Operate the microdialysis pump at a rate of 1 µL/min to inject ACSF into the probe until the surface of the probe is slightly moist. Immerse the probe in heparin sodium injection solution for subsequent use.
   ​NOTE: If a big stream of the ACSF falls from the semipermeable membrane of the probe, as seen with the naked eye under gravity, replace the probe with a new one.

3. Collection of HECF from the awake rat

1. Insert the brain microdialysis probe into the probe catheter and place the rat in a chamber (height: 360 mm; diameter: 400 mm) with padding to make sure the rats are free to move around.
2. Connect the pipeline, microsyringe pump, and brain microdialysis probe. Harness the rat via the hole at the top of the rat restraint device and the stainless-steel swivels.
3. Turn on the multi-channel swivel controller to avoid intertwining the microdialysis pipelines during the free movement of the rat. Turn on the microsyringe pump and pump the ACSF at a rate of 1 µL/min. Collect HECF periodically after a 60 min equilibration of the microdialysis HECF collection system.
4. Ensure that the flow rate of the HECF samples in the refrigerated fraction collector is consistent with ACSF infusion. Collect 20 µL of HECF and automatically change to the next sampling tube. Take care to check whether the probe membrane is damaged when inserting the probe.

4. Measurement of the osmotic pressure for the HECF

1. Turn on the osmometer and log in to the detection system. Click on the Cal button on the touch screen and click on the Res button on the page to clear the previous calibration memory.
2. Install a 1.5 mL tube containing 100 µL of pure water without bubbles on the measuring head. Pull the measuring head to the bottom of the cold hydrazine container.
3. Enter the sample number 0 on the touch screen and confirm to test. Quickly dip the diode needle into the sample tube, and then quickly pull it out to induce crystallization of the sample at a temperature of -6.2 °C.
4. Wait for the screen to display: Push measure head up and click on Cal and Cal 0 in turn to calibrate. Perform measurements with a 300 mOsm calibration solution and measure the osmotic pressure of the HECF samples as described above.
   ​NOTE: Wipe the measuring head with a soft paper towel after calibration or measurement. The HECF samples with no bubbles should be mixed well.

5. Maintenance of the microdialysis system and devices after sampling

1. Take out the brain microdialysis probe from the probe catheter after sampling termination. Immerse the probe in deionized water and lavage with deionized water for 12 h to remove stranded salt depositions from the pipeline and the probe.
2. Remove the probe to place into a 0.05% trypsin solution at 4 °C. Dry the pipelines in an air-dry oven at 25 °C and store them at room temperature.
   ​NOTE: The microdialysis probes are expensive and this step can increase the reusability of the probes. Proteins that adhere to the probe surface can be digested by trypsin solution to prevent the probe membrane from being blocked by proteins, and trypsin had no effect on the probe material.

6. Animal treatment after sampling

1. After sampling, painlessly euthanize the rats by making them inhale 1.5% isoflurane, followed by an overdose of 5% isoflurane in accordance with animal ethics.