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1 b a Magnet Illumination Coverslips MPs Objective 2575 µm 1875 µm 1575 µm 1075 µm 875 µm 545 µm 20µm mm Field Induction (Gauss) Distance (mm) Distance (mm) Mag. Force (pn) d c µm 0.3 mm 0.5 mm 1 mm 1.5 mm 2 mm 3 mm Distance (mm) Magnetization (emu/g) e Applied Field (x10 A/m) Supplementary Figure 1 Properties of magnets and MPs. a, Illustration of the setup for in vitro tests of the effect of magnetic force on 180-nm MPs within a 100-μm film of solution (sandwiched by two coverslips). b, Dark-field imaging of particle aggregation as a function of distance away from the film. The distance between the 0.3-mm magnet and MPs is marked on each image. Each image was recorded after brief stabilization of particle density. c, Experimentally measured magnetic field induction profiles and scaled theoretical calculations. The field induction was measured at mm away from the magnet. To approximate the field strength closer to the magnet, we scaled the theoretical curves to match the experimental curves. Inset, six magnets of different size (0.3 3 mm) were utilized. d, Estimates of magnetic force computed from the magnetic field gradients. Measurement of the magnetization of 180-nm MPs using an alternating gradient magnetometer. e, Superparamagnetic particles with low remanence were magnetized and demagnetized by the application/removal of an external magnetic field.
2 Supplementary Figure 2 Toxicity test for the 180-nm MPs. a, HEK293T cells were incubated with 1 or 2 mg/ml MPs for 4 h and then washed with PBS. Images were taken at 0 h (before MPs were incubated with cells), 4 h (when MPs were removed after 4-h incubation), and 24 h (20 h after MPs were removed). For the 1 mg/ml and 2 mg/ml groups, a small amount of MPs remained in wells after washing. b, Summary of data for cell survival (n = 3 4 wells per group). There was no significant difference between different groups (n.s., P > 0.05, two-tailed unpaired t-test). Error bars indicate s.e.m.
3 Supplementary Figure 3 In vitro toxicity test for 180-nm MPs. a, Both images were acquired at day 4 (d4). Ctrl, the group with no MPs, left panel; MP, the group with 2 mg/ml MPs incubated in medium for 24 h before washing, right panel. After fixation, nuclei of astrocytes were stained with Hoechst (HO342). b, Summary of data for astrocyte survival (n = 4 wells per group). There was no significant difference between the Ctrl and the MP group (n.s., not significant, P > 0.05, two-tailed unpaired t-test). Error bars indicate s.e.m.
4 Supplementary Figure 4 MPs in live mice. a, b, Coronal T2-weighted magnetic resonance images before and after injection of MPs in mouse 1 (a) and 2 (b). Images were acquired with the Aspect Imaging M2TM 1.0 T system using a to 45-MHz, 35-mm diameter RF mouse coil. Fast Spin Echo sequence was performed with the following parameters: repetition time = 6000 ms; effective echo time = 103 ms; field of view mm 2 ; data matrix = ; averaging = 2; slice = 1 mm. An obvious magnetic resonance imaging contrast change was observed in the liver/spleen after tail-vein injection of MPs.
5 Supplementary Figure 5 Measurement of basic physiological properties of mice. We measured respiratory rate, body temperature, heart rate, SpO 2, and body weight before (baseline) and after injection of PBS (control, n = 7 mice) or MPs (100 μg/g body weight, n = 7) into the tail vein. Four time points were assessed for each group: Before injection (Before), 3 h, 24 h (1d), and 5 days (5d) after injection. There was no significant difference for any of the measurements between the PBS and MP groups at any of the time points (n.s., not significant, P > 0.05, twotailed unpaired t-test). Error bars indicate s.e.m.
6 Supplementary Figure 6 Measurement of ECO 2 and blood glucose level. a, b, The concentration of carbonate and glucose were measured before and after we injected MPs (~100 μg/g body weight) into adult mice (ECO 2, n = 5 9 mice for each time point; glucose, n = 5 12 mice). The baseline (d1) was measured at day 1 before we injected MPs via tail vein. The number of mice used for each time point is shown in the histogram. There was no significant difference between the baseline and the values from other time points (n.s., not significant, P > 0.05, two-tailed unpaired t-test). Error bars indicate s.e.m.
7 Supplementary Figure 7 In vivo toxicity test for 180-nm MPs. a, PBS (control, 1μl) and MPs (1 μl, 10 μg/μl) were injected into the hippocampal region of adult CX 3 CR1-GFP knock-in mice. There was no significant difference in microglial morphology and density between the PBS and MP groups (n = 3 mice per group). Green, enhanced GFP fluorescence in microglia in CX 3 CR1-GFP mice. Inj. site, locations where PBS or MPs were injected. b, The image from the location close to the location in (a) in the hippocampus. c, Summary of results for microglial density as shown in (b). There was no significant difference in microglial density between the PBS and MP groups (n.s., not significant, P > 0.05, two-tailed unpaired t-test). Data were obtained from 7 brain sections of 3 mice. Error bars indicate s.e.m.
8 Supplementary Figure 8 Toxicity test for the 180-nm MPs in vivo. a, PBS (control, 1 μl) and MPs (1 μl, 10 μg/μl) were injected into the hippocampal region of adult Thy1-TdTomatotg mice. Wide-field images (WF) were taken with a dissection microscope. Other images were taken with a confocal microscope. b, Representative images of spines in apical dendrites of CA1 neurons. c, There was no significant difference (n.s., P > 0.05, two-tailed unpaired t-test) in the morphology of hippocampal neurons (n = 3 mice) and spine density in apical dendrites (PBS, n = 10 neurons; n = 13 neurons, MP) between the PBS and MP groups. Red, tdtomato (Tdt) signal from neurons in Thy1-tdTomato. Blue, nuclei stained with Hoechst (HO).
9 Supplementary Figure 9 Blood vessels in ischemic regions. Blood vessels from a non-ischemic area (left panels, control region) and a region that underwent MP-mediated ischemia (right panels, ischemic region). The results were from an adult mouse brain (>P50, 40-min occlusion followed by 72-h reperfusion before fixation). Blood vessels were stained with anti-laminin (green). Disrupted blood vessels (magenta arrows) were observed in the ischemic region. The cortical layer structure was disrupted in the ischemic region (right bottom). The cortical layers I III are indicated. Blue indicates DAPI-stained nuclei.
10 Supplementary Figure 10 Different segments of blood vessels in the brain of NG2DsRedBACtg mice. a, SMCs in arteries/arterioles with a ring-shaped morphology. b, Pericytes in veins with multiple-process morphology. c, Both pericytes in capillaries and NG2 glial cells were DsRed + (red fluorescence) in this transgenic line.
11 Supplementary Figure 11 SMCs and pericytes in blood vessels. SMCs (upper) and pericytes (middle and lower) in fixed brain sections from an NG2DsRedBACtg mouse. Green fluorescence indicates staining with anti-αsma. Red, DsRed fluorescence from the NG2DsRedBACtg mouse. Blue indicates DAPI-stained nuclei.
12 Supplementary Figure 12 Loss of SMCs in the brain with 2-h MCAO in juvenile mice. a, b, SMCs were lost in the ischemic region (b) but not in the control region (contralateral hemisphere) (a) of a juvenile mouse after a 2-h MCAO. SMCs were labeled with DsRed in NG2DsRedBACtg mice (red). Arrows indicate locations in which DsRed fluorescence was lost; nuclei were stained with Hoechst (blue). c, There was a significant difference in SMC loss between the control region and ischemic region in juvenile mice (n = 7) but not adult mice (n = 5) under MCAO. *P < 0.05; **P < 0.01; ***P < 0.001, two-tailed unpaired t-test). Error bars indicate s.e.m.
13 Supplementary Figure 13 Staining of SMCs in arteries/arterioles in the brain after MCAO. The signal for SMA was weak in locations lacking DsRed fluorescence (i.e. likely SMC loss, arrows) after a 2-h MCAO in a juvenile mouse.
14 Supplementary Figure 14 Time-lapse imaging of SMCs under ischemia induced by MPs. a, b, Intensity of DsRed fluorescence at different time points after MP-mediated occlusion. Loss of DsRed fluorescence occurred within 5 min in regions 4 and 5. c, Blebbing (white arrows) and formation of intracellular vacuoles (blue arrows) were observed in SMCs after occlusion.
15 Figure Number Mouse Age Body Weight (g) Magnet (mm) Occlusion Reperfusion Fig.1e Adult (3 mon) min Fig.1f Adult (3 mon) min Fig.2a d Juvenile (P15 25) N/A 1.0 & min Fig.2e,f,g Juvenile & adult N/A min Fig.2h i Adult (>3 mon) min Fig.3a Perinatal (P3) min Fig.3b Adult (P>50) N/A Fig.3c,d Juvenile (P15) N/A 1 90 min Fig.3f Juvenile & adult 6 11 & min Fig.3g Juvenile (P15 25) min 2 125min Fig.3h Adult (P>50) 1 2 h 6d Fig.4a Adult (P>50) , 1, 5, 48h Fig.4b Adult (P>50) min 3d Fig.4c Adult (P>50) min 3d Fig.5b Adult (P68) min Fig.5d Adult (P ) N/A min Fig.5a c,e g Juvenile (P15 25) min Fig.5h Junvnile (P15 25) min Suppl. Fig. 4 Adult (>P50) 25 g N/A Suppl. Fig. 5 Adult (2 mon) N/A Suppl. Fig. 6 Adult (2 3 mon) N/A Suppl. Fig. 7 Adult (6 12 mon) N/A N/A Suppl. Fig. 8 Adult (6 12 mon) N/A N/A Suppl. Fig. 9 Adult (>P50) min 3d Suppl. Fig.14 Juvenile (<P28) min
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