All animal experiments were performed in compliance with the Guidelines for the Care and Use of Laboratory Animals at Osaka University School of Medicine and the Guidelines of the NIH for the Care and Use of Laboratory Animals. Three- to four-month-old male mice (C57BL6/J) weighing 20–25 g were used in the study (obtained from CLEA Japan). Animals were maintained at room temperature (25 ± 2 °C) under a standard 12-h/12-h light-dark cycle, with free access to water and food.
Surgical procedure for CDP implantation
The CSF collection tubing for CDP (M025V-100, EICOM, Japan), made of polyurethane (0.3 mm i.d.), was cut to a length of 3 cm via a razor blade. The CSF collection tubing was connected to the FEP tube with joint (JF-10, #800160, EICOM, Japan), which was connected to a peristaltic pump (ERP-10, #600100, Eicom, Japan) or microsyringe pump (ESP-64, Eicom, Japan). The tubing was filled with distilled water prior to surgery.
Mice were anesthetized with an intraperitoneal injection of a combination of domitor (0.75 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg), and then mounted on a stereotaxic frame (SR-5M-HT, NARISHIGE, Japan), with a heating pad placed underneath the body. The mouse is laid down so that the head approximately forms an 80-degree angle with the body, secured by using the head adaptor. The surgical area was disinfected with iodine and covered by a surgical drape. Using straight-tip surgical scissors, an incision (20 mm) in the skin was made at the center of the neck. Under the dissection microscope, the subcutaneous tissue and muscle layers were separated by blunt dissection with forceps and fine-tipped cotton swabs to expose the AOM of CM.
A small hole was made in the center of the AOM by shallow centesis using a 30G needle, with caution taken to avoid damage to brain tissue and blood vessels. After confirming the CSF flowing out of the puncture due to cerebrospinal pressure, the tip of the CSF collection tubing was attached to the membrane by folding the tubing with a stereotactic arm so that it covered the small hole made on the AOM. Then the CSF was withdrawn via a peristaltic pump at a flow rate of 20 μl/hour, which visually confirmed that the CSF was being drained into the cannula. After confirming this, the flow rate was decreased to 10 μl/hour.
The CSF collection tubing, AOM, and surrounding bone structures, including the occipital crest and anterosuperior border of the atlas, were fixed with 4-META/MMA-TBB resin (Super-bond C&B, Sun Medical, Japan). After confirming that the CSF continued to be drained and the cement was completely secured, the flow rate was further decreased to 4 μl/hour.
The CSF collection tubing was disconnected from the FEP tube, and the outlet was plugged. The skin was aligned and sutured using 7-0 silk, then a mixture of analgesic, sugar, and antibacterial agents were administered intraperitoneally. The mouse was left on top of the heating pad to maintain its body temperature until it awoke. The mice were then carefully transferred to a recovery home cage. Furthermore, they were allowed at least four days for tissue recovery before starting continuous CSF collection.
Continuous CSF collection via CDP
Four days after the implantation of CDP, the mice were tethered with the sensor-integrated balance arm and placed into the movement-response caging system (MD-1409, BASi, USA as described previously (17). The sensor detects the rotation of the animal and turns the cage in the opposite direction, which allows unrestricted movement of the animals without applying pressure to the probe assembly. The CSF collection tubing (M025V-100, Eicom, Japan) from the CDP is connected to a roller pump (ERP-10, Eicom, Japan) or syringe pump (ESP-32, Eicom, Japan) to withdraw CSF at a constant flow rate. The movement-response caging system was placed in the sound-attenuating box (Natsume Seisakusho, Osaka, Japan) to control environmental conditions; the mice were maintained at room temperature (25 ± 2 °C) under a standard 12-h/12-h light-dark cycle, with free access to water and food. The locomotor activity of the mice during the experiment was measured by an infrared light beam crossing system as described below.
A single-collection technique for mouse CSF via CM puncture
A conventional single-collection technique for mouse CSF was performed as previously described, with minor modifications (10). Mice were anesthetized with an intraperitoneal injection of a combination of domitor (0.75 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg), then placed on a warmed surgical platform. Under the dissection microscope, the subcutaneous tissue and muscles are separated to expose the CM. CSF was collected by puncturing the AOM with a 30G needle and aspirated with a P20 pipettor.
Chronic CMc method
For the chronic CMc experiment, implantation of a catheter into the cisterna magna in mice was performed as described earlier (12). Three days after CMc surgery, the mice were euthanized, and their brains were harvested for histology to evaluate the tissue damage due to the intrathecally inserted cannula.
Assessment of blood contamination and measurement of electrolytes in CSF
A high-sensitivity spectrophotometry method was used to measure blood contamination in the collected CSF, as described previously (10). Levels of hemoglobin in the collected CSF, released by the hypotonic freeze-thaw method, were quantified by measuring the absorbance at 417 nm on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies/Thermo Scientific, Wilmington, DE, USA). We used clear CSF spiked with increasing amounts of whole blood (0.01%, 0.1%, and 1%) as references. Pooled CSF was centrifuged at 2,000 g for 30 seconds to remove any red blood cells, and the supernatant was used for clear CSF. The concentrations of electrolytes (sodium, potassium, and chloride) in the collected CSF were measured by a blood gas analyzer (Radiometer ABL 700, Radiometer Medical, , Denmark)
Biosensor measurements of glucose and lactate in CSF
Enzyme-based biosensors were used for real-time monitoring of glucose and lactate levels as previously described (24, 33). In this study, the distal end of the electrodes of glucose and lactate biosensors (Part #7004-80‐Glucose and #7004‐80-Lactate; Pinnacle Technology, U.S.A.) were inserted in the biosensor port (Eicom, Japan), where the biosensors were exposed to the continuously collected CSF. All biosensors were calibrated in vitro prior to implantation. Mice were given a single dose of an intraperitoneal injection of glucose (2 g/kg body weight) during biosensor measurements of CSF glucose and lactate.
Brains were fixed in 4% PFA for 24 hours at 4 °C, incubated for 2 days in 30% sucrose in PBS and embedded in a Tissue-Tek O.C.T. Compound (Sakura Finetek Japan Co., Ltd.), and then the sagittal and coronal sections of 8 μm thickness were cut on a cryostat and mounted on glass slides. Brain sections were stained with hematoxylin and eosin (H&E) to evaluate the tissue integrity and damage due to CSF collection. For immunostaining, sections were permeabilized with 0.5% Triton X-100 in PBS, blocked in 5% BSA in PBS, and immunostained with rabbit anti-GFAP antibody (1:200, PRB-571C, BioLegend) and mouse anti-NeuN antibody (1:200, MAB377, Merck) in 1% BSA in PBS at RT overnight. Sections were washed thoroughly in PBS, and then immunoreactions were visualized by fluorescent secondary antibodies. Sections were mounted using VECTASHIELD medium (H-1000, Vector Laboratories). We used the following secondary antibodies: Alexa 488-conjugated goat anti-rabbit IgG antibody (1:200, ab150077, abcam) and Alexa 594-conjugated goat anti-mouse IgG antibody (1:200, ab150116, abcam). Fluorescent images were captured with a fluorescence microscope (BZ-9000, Keyence, Japan) equipped with a digital camera, using the same fluorescence settings in all cases. The pixel intensities of the fluorescent signal were analyzed and quantified using National Institutes of Health Image software. For GFAP burden analyses, data was reported as the percentage of the labeled area captured (positive pixels) divided by the full area captured (total pixels).
Drug and tracer injection via CDP
Intrathecal injection of drug and CSF tracer injection was performed by using a 30G Hamilton microsyringe, which was connected to a CDP via CSF collection tubing. The dead volume of CSF collection tubing was approximately 0.6 μl. A single dose of contrast agent (5 μl in 10 seconds) was injected via CDP, followed by sequential micro-CT imaging (R_mCT2, Rigaku Corp., Japan). Concentrations of contrast agents at each brain region at each time point were determined based on the signal intensity, which were used for calculating metabolic parameters such as t1/2 and Tmax. For histological assessment, a single dose of a water-soluble nuclear-staining dye (Hoechst 33342, invitrogen, USA) was injected via the CDP (50 μg/5 μl in 10 seconds). The brain was harvested at 60 min. Coronal sections were immunostained with an anti-NeuN antibody as described above, and fluorescent images were captured with a fluorescence microscope (BZ-9000, Keyence, Japan). For behavioral assessment of an intrathecally delivered drug, an anesthetic agent (midazolam, Maruishi Pharmaceutical, Japan) was acutely injected via CDP in the free-moving mice (25 μg/5 μl in 1 sec by a syringe connected to the CDP), while locomotor activities were measured by an infrared light beam crossing system as described next. Following a baseline measurement for 5 min, an anesthetic agent was acutely delivered, and locomotor activity recording continued until 10 min.
Behavioral and locomotor measurements using activity monitoring system
Spontaneous locomotor activity of mice during the continuous CSF collection or intrathecal drug delivery experiment was monitored in an open-filed cage (43 cm to 43 cm) with high-density arranged infrared sensors (Scanet, MV-40, Melquest, Toyama, Japan).(23, 34) The infrared sensors are distributed in all directions parallel to the floor (37.5 mm from the floor). The mice were allowed to move freely on the floor. Mice movements were detected by infrared sensors and recorded every 0.1 s.
All data were expressed as the mean ± s.e.m. Two-group comparisons were performed by an unpaired t-test, unless stated otherwise. Comparison among three or more groups was performed by analysis of variance and the Tukey-Kramer test, unless stated otherwise. P values < 0.05 were considered significant.