The authors declare that all supporting data are available within the article.
2.1. Fabrication of cell-implantable radiopaque hydrogel microfibers
Cell-implantable radiopaque hydrogel microfibers were prepared using a method similar to that reported previously[25]. A double coaxial laminar flow microfluidic device[24, 25, 27] was used to fabricate fiber-shaped hydrogels containing X-ray-opaque particles. This device comprised glass capillaries with a tapered tip, square glass capillaries, and connectors made of resin. The glass capillaries with an outer diameter (OD) of 1.0 mm and an inner diameter (ID) of 0.6 mm (G-1; Narishige, Tokyo, Japan) were sharpened by a puller (P-10; Narishige). The tips of these glass capillaries were cut using a micro-forge (EG-44; Narishige) to a tip diameter of 600 µm. Square glass capillaries with an OD of 1.4 mm and an ID of 1.0 mm (8100 − 100; VitroCom, NJ, USA) were used to fix the inner processed glass capillaries. Connectors were fabricated using a 3-dimensional printer (AGILISTA; Keyence, Osaka, Japan). Finally, the glass capillaries and connectors were assembled on a slide glass (S2142; Matsunami Glass, Osaka, Japan).
For pre-gel solutions for microfiber fabrication, 1.5% (w/w) sodium alginate solution (194-13321; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) was prepared as the core flow. For the shell, 2.5% (w/w) sodium alginate solution (194-13321; Wako) mixed with 30-µm diameter zirconium dioxide particles (NZ30; Niimi, Aichi, Japan) was prepared. Next, a 100 mM barium chloride solution (B0750; Sigma Aldrich, St. Louis, MO, USA) was prepared for sheath flow. Microfibers were formed according to the following procedure. Three syringes were filled with the 1.5% (w/w) sodium alginate solution for the core flow, with the 2.5% sodium alginate solution containing zirconium dioxide particles for the shell flow, and 100 mM barium chloride solution for sheath flow, respectively, in a refrigerator (4°C). All syringes were connected to the inlets of the double coaxial microfluidic device via Teflon tubes (JR-T-4183-M10; Shimadzu Corp., Kyoto, Japan) then set to syringe pumps. These syringe pumps sequentially started to infuse the core flow rate of 75 µL/min, shell flow rate of 225 µL/min, and sheath flow rate of 2000 µL/min into the microfluidic device, creating a microfiber with a diameter of 0.4 mm and a zirconium dioxide concentration of 60% (w/w). The ejected microfiber was collected in ultrapure water and cut to a length of 1 mm with scissors (Fig. 1A).
2.2. Animal model and protocol
Male normotensive adult Sprague-Dawley rats (n = 14; 13 weeks old; median body weight, 391 g; IQR, 381–415 g on the day of surgery) were obtained from Nihon SLC (Japan SLC, Shizuoka, Japan) and 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, Tokyo, Japan). Our study was approved by the Institutional Animal Care and Use Committee of Jikei University School of Medicine (protocol numbers: 2016 − 105 and 2019-054). 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. Furthermore, this study was carried out in compliance with ARRIVE guidelines. This study did not need a control group because the study involved making a stroke model. Anesthesia was maintained with 1–3% isoflurane through a vaporizer for small experimental animals and a facial mask. Body temperature was maintained before surgery at 36–37.5°C using a multi-panel heater (Vivaria, Osaka, Japan). As previously reported, the body of the animal (including the entire tail) must be kept warm[28].
Utilizing the “Ohta Method”[29], a simple method for reaching the major cerebral arteries in rats using a microcatheter and microfibers is shown in Fig. 1B. The same method reported previously was used to create the stroke model[30]. The polyamide microcatheter (ID 0.42 mm, OD 0.55 mm; Kaneko Cord, Tokyo, Japan) was first flushed with heparinized physiological saline. The rat was then placed supine with its tail outstretched. An indwelling needle (venous indwelling needle for humans, 22 G, SR-OT2225C; Terumo, Tokyo, Japan) was inserted through the caudal ventral artery. After confirming arterial blood backflow from the indwelling needle, a microcatheter and wire (OD 0.4 mm, FGW16-AG18S30; Toray Medical, Tokyo, Japan) were inserted. The wire and microcatheter were guided from the caudal ventral artery to the abdominal aorta, aortic arch, and left common carotid artery (CCA) using the digital subtraction angiography (DSA) unit (Artis Zee, Siemens, Germany). Left CCA angiography was then performed, in which 0.1 ml of contrast medium (Iohexol; Daiichi Sankyo, Tokyo, Japan) at half concentration was injected from the microcatheter under DSA. Contrast images were overlaid to improve visualization, and the microcatheter was guided to the origin of the left ICA. Selective angiography of the left ICA could then detect the left anterior cerebral artery (ACA), MCA, and PCA (Fig. 1C). The syringe was filled with heparinized physiological saline. The capillary tube was filled with a 1-mm-long radiopaque hydrogel microfiber with a diameter of 0.4 mm (Fig. 1A). The capillary tube was then connected to the catheter and inserted into the tail artery. The microfiber was delivered by extruding the microfiber through slow injection of heparinized physiological saline. At this time, the position of the microfiber was monitored in real time under DSA. This procedure ensured that the microfiber completed embolization to the ACA-MCA bifurcation (Fig. 1C and Supplemental Video). Five milliliters of physiological saline was injected subcutaneously to prevent dehydration after model preparation. The time from puncture to model creation, the amount of contrast agent administered and blood loss were measured. The frequency of hemorrhagic complications, including subarachnoid hemorrhage, was also evaluated.
2.3. Neurological deficit score and body temperature
Neurological function was evaluated before and at 3, 6, and 24 h after induction of ischemia using neurological deficit score, a 0–5-point scale described previously[31, 32]. Scores were defined as: 0 = no neurological deficit; 1 = failure to extend right forepaw fully when lifted by tail; 2 = circling to the right; 3 = falling to the right; 4 = no spontaneous walking or in a comatose state; 5 = dead. Body temperature was measured with a rectal probe just after creation of the stroke model and before each neurological evaluation.
2.4. X-ray fluoroscopic analysis
X-ray fluoroscopy was performed 0, 3, 6, and 24 h after induction of ischemia to determine whether spontaneous migration of the radiopaque hydrogel microfiber had occurred.
2.5. Magnetic resonance imaging (MRI) analysis
MRI was performed at 3 and 6 h after model creation, using a 9.4-T BioSpec 94/20 system (Biospin GmbH, Ettlingen, Germany), a transmitting coil with a 72-mm inner diameter and a rat brain size 4 channel receiver coil. Diffusion-weighted imaging (DWI) was performed using the pulse field gradient spin-echo pulse sequence[33]. The following parameters were set: repetition time, 3000 ms; effective echo time, 32.06 ms; flip angle, 90°; field of view, 20×20 mm2; matrix size, 80×80; image resolution, 250×250 µm; number of slices, 30; slice thickness, 1 mm; number of DW directions, 3 (each with a b-value of 1000 s/mm2); b, 0 s/mm2; motion-probing gradient duration, 10.0 ms; and motion-probing gradient interval, 15.8 ms. Total DWI scan time was 8 min 48 s. T2-weighted imaging (T2WI) was performed using rapid acquisition with a relaxation enhancement pulse sequence[34]. The following parameters were set: repetition time, 5000 ms; effective echo time, 45.0 ms; flip angle, 90°; RARE factor, 8; field of view, 20×20 mm2; matrix size, 200×200; image resolution, 100×100 µm; number of slices, 30; and slice thickness, 1 mm. Total T2WI scan time was 4 min 10 s.
To acquire MRI data, 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 scanning to manage the physical condition of the animal. Volumetric measurements of the whole brain and ischemic regions were measured from images obtained during MRI. The analysis software used was BrainSuite18a (David W. Shattuck, Ahmanson-Lovelace Brain Mapping Center at the University of California). Regions were extracted and volumes were measured using the masking tool in the software. Boundaries were determined for the ischemic region based on discussions among three neurologists and neuroscientists with more than 10 years of experience in the field who were blinded to the details of the surgical procedures.
2.6. Postmortem analysis
At 24 h after embolization, animals were deeply anesthetized and euthanized. If the rat died before 24 h had elapsed, survival time was recorded and an autopsy was performed immediately. The brain was removed and sectioned coronally into 6 slices (thickness, 2 mm) from the olfactory bulb to the cerebellum, then stained with 2% 2,3,5-triphenyl tetrazolium chloride (TTC) for 20 min. Using image analysis software (ImageJ, version 1.53a; National Institutes of Health, MD, USA), the non-infarcted area of the ipsilateral hemisphere and area of the contralateral hemisphere were calculated on the rostral and caudal sides, respectively.
Infarct volume in each section (mm3)
= (area of contralateral hemisphere - non-infarcted area of ipsilateral hemisphere) on rostral side (mm2) × 1 (mm)
+
(area of contralateral hemisphere - non-infarcted area of ipsilateral hemisphere) on caudal side (mm2) × 1 (mm)
The sum of each section volume was considered as the total volume of infarction[35, 36]. Boundary determination for the infarct volume was based on discussions among three neurologists and neuroscientists with more than 10 years of experience in the field who were blinded to the details of the surgical procedures.
2.7. Statistical analysis
We performed univariate analyses to determine whether changes occurred over time in ischemic lesion, body temperature, or neurological deficit score. Friedman’s test was used to assess differences in ischemic volume (DWI and TTC) between 3, 6 and 24 h, body temperature between 0, 3, 6, and 24 h, and neurological function between before and 3, 6 and 24 h after MCAO. The Wilcoxon signed-rank test was then used to assess differences in ischemic volume on T2WI between 3 and 6 h after MCAO. In addition, Mann-Whitney's U test was used to examine whether differences existed in infarct volume after 24 h with or without migration of the microfiber. Values of p < 0.05 were considered significant. All statistical analyses were performed using IBM SPSS Statistics version 27 software (IBM-Armonk, New York, NY).