The authors declare that all supporting data are available within the article.
Animal model and protocol
Male normotensive adult Sprague Dawley rats (n = 8; 11-27 weeks of age; 366-587 g on the day of surgery) obtained from Nihon SLC (Japan SLC, Inc. Shizuoka, Japan) were housed in polycarbonate cages under temperature-controlled conditions (temperature: 24-25°C; relative humidity: 50-60%) with a 12-h light-dark cycle. All rats had free access to water and pelleted food (CE-2, CLEA Japan, Inc., Gifu, Tokyo, Japan). Our study was approved by the Institutional Animal Care and Use Committee of the Jikei University School of Medicine (protocol number: 2016-105). All procedures were conducted according to the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions issued by the Japanese Ministry of Education, Culture, Sports, Science, and Technology. This study doesn’t need the control group because of making stroke model study. Anesthesia was maintained with 1-3% isoflurane through a vaporizer for small experimental animals and a facial mask. Body temperature was maintained during surgery at 36-37.5°C using a multi-panel heater (Vivaria, Osaka, Japan). The animal’s body, including the entire tail, must be kept warm, as previously reported.20 No preparation, such as shaving, disinfection, or antibiotics, is required.
Major arteries of rat with microcatheter and zirconia ball and a simplified operation scheme are shown in Fig.1. The polyamide microcatheter (inner diameter (ID) 0.42 mm, outer diameter (OD) 0.55 mm, Kaneko Cord, Tokyo, Japan) was first flushed with heparinized physiological saline. The rat was placed in a supine position with a stretched tail. Indwelling needle (venous indwelling needle for human, 22 gauge, SR-OT2225C, TERUMO, Tokyo) was inserted through the ventral midline artery approximately 5 cm from the root of the tail of the rat (Fig. 2A). After confirmation of arterial blood backflow from the indwelling needle, a microcatheter and wire (OD 0.4mm, FGW16-AG18S30, Toray Medical, Tokyo, Japan) were inserted as long as no resistance was felt. Upon resistance, the insertion of the microcatheter was stopped immediately, and the position of the microcatheter was confirmed using fluoroscopic imaging. The wire and microcatheter were guided from the caudal ventral artery to the abdominal aorta (Fig. 2B), the aortic arch, and the left common carotid artery (CCA) (Fig. 2C) using fluoroscopic imaging. Then, the wire was carefully removed so as not to change the position of the microcatheter. The contrast media (Iohexol, Daiichi Sankyo, Tokyo, Japan) at half concentration was rapidly injected from the microcatheter. Then, left CCA angiography was performed, in which 0.1 ml of the contrast media was injected using a digital subtraction angiography unit (Artis Zee, Siemens, Germany; Fig. 2D). The bifurcation of the ICA branch was visualized by tilting the C-arm at an LAO angle of 80° (Fig. 2D). The contrast images were overlaid for improving visualization, and the microcatheter was guided to the branch of the left ICA. Left ICA selective angiography could then detect the left anterior cerebral artery (ACA), MCA, and PcomA (Fig. 3A). Then, one zirconia ball (zirconium dioxide, 0.4 mm in diameter; Nikkato, Tokyo, Japan. Fig. 4A) was advanced in the microcatheter by slow injection of approximately 0.1 ml of heparinized physiological saline (Supplemental File - Videos 1 and 2), and the ACA-MCA bifurcation was selectively embolized (Figs. 3B, 4B). The zirconium dioxide does not affect the MRI because of non-magnetic material. The high visibility of zirconia ball during fluoroscopy was found to be comparable to that of wire. The microcatheter was inserted into the right CCA and contrasted to confirm that the left ACA had retrograde blood flow from the right ACA and only obstructed the left MCA in the frontal view (LAO angle 0°) (Fig. 3C). The time from the puncture to model creation was measured. The plastic outer indwelling needle was fixed with sealing film (Parafilm, Bemis, United States of America) and gaffer tape and was not removed until 24 hours after the embolization. Then, cerebral angiography was performed to confirm whether spontaneous recanalization had occurred. No postoperative pain relief was required.
Cerebral angiography 24 hours after the creation of the stroke model
Cerebral angiography was performed 24 hours after the creation of the stroke model to determine whether spontaneous recanalization had occurred. Cerebral angiography could be performed in the same way as when the model was created without re-puncture by using the plastic outer indwelling needle that was left behind.
Magnetic resonance imaging (MRI) analysis
MRI was performed using a 9.4-T BioSpec 94/20 (Biospin GmbH, Ettlingen, Germany) device and a transmitting coil with a 72-mm inner diameter and a rat brain size 4 channel receiver coil immediately before the rats were euthanized. T2-weighted imaging and MR angiography were performed using the time of flight theory. T2-weighted imaging was performed using the rapid acquisition with a relaxation enhancement pulse sequence21. The following parameters were set: repetition time: 5000 ms, effective echo time: 42.0 ms, flip angle: 90°, RARE factor: 8, field of view: 20×20 mm2, matrix size: 200×200, image resolution: 100×100 μm, and slice thickness: 800 μm. The total T2-weighted imaging scan time was 6 minutes and 15 seconds. MR angiography was performed using a three-dimensional fast low angle shot pulse sequence22. The following parameters were set to achieve the time of flight effect: repetition time: 14 ms, echo time: 2.28 ms, flip angle: 8°, number of average: 6, field of view: 20×18×22 mm2, matrix size: 166×148×180, and image resolution: 122×122×122 μm. The total scan time of the three-dimensional fast low angle shot pulse sequence was 36 minutes 20 seconds. MR angiography was reconstructed using the maximum intensity projection method in ParaVision version 6.0.1 software (Bruker, Ettlingen, Germany). To acquire MRI data, the rats were scanned in the prone position on an imaging stretcher and administered a mixture of air and 1.5-3.0 % concentrated isoflurane (Abbott Laboratories, Abbott Park, IL, USA). Respiration was regularly monitored during the scanning to manage the animal’s physical condition.
Postmortem analysis
Twenty-four hours after embolization, the animals were deeply anesthetized and euthanized. The brains were removed and were sectioned coronally into 6 slices (thickness, 2 mm) from the olfactory bulb to the cerebellum and then stained with 2% 2,3,5-triphenyl tetrazolium chloride (TTC) for 20 minutes. Using image analysis software (ImageJ, version 1.53a), the non-infarcted area of the ipsilateral hemispheres and the areas of the contralateral hemisphere were calculated on the rostral side and caudal side, respectively.
Infarct volume on each section (mm3) = (areas of the contralateral hemisphere - the non-infarcted area of the ipsilateral hemisphere) on the rostral side (mm2) × 1 (mm) + (areas of the contralateral hemisphere - the non-infarcted area of the ipsilateral hemisphere) on the caudal side (mm2) × 1 (mm)
The sum of each section volume is considered to be the total volume of infarction23,24. A postmortem analysis was performed by one researcher (H.M.), who was blinded from details of the surgical procedures.