Supporting Information - PNAS...AB 200 μm Baseline After irradiation 350 μm C Baseline After...
Transcript of Supporting Information - PNAS...AB 200 μm Baseline After irradiation 350 μm C Baseline After...
Supporting InformationChoi et al. 10.1073/pnas.1018790108SI Materials and MethodsHistology.Mice were killed under deep anesthesia by transcardialperfusion with PBS, followed by fixation with 4% formalin inphosphate buffer. Brains were removed, immersed in 4% for-malin in phosphate buffer overnight, and then transferred toa 30% sucrose solution for at least 3 d. Target regions werenavigated by stereotaxic coordinates and fluorescence, followedby sectioning into 30-μm coronal sections on a freezing slidingmicrotome. The sections were stained using the Nissl methodor immunostaining and mounted on glass slides for microscopicevaluation.
Dorsal Skinfold Chamber Model. A small dorsal skinfold chamberkit (SM100) was purchased from APJ Trading, and surgery wasperformed as described previously (1). In brief, mice were an-esthetized with an i.p. injection of ketamine–xylazine, the dorsalskin was extended, and two mirror-image titanium frames weremounted. One layer of skin was excised, leaving the striatedmuscle of the opposite side intact, and then replaced with a glasscoverslip mounted into the frame.
In Vivo Skull Imaging Preparation. After anesthesia with an i.p.injection of ketamine-xylazine, the scalp was removed, and themouse head was fixed to a stereotaxic frame.
In Vivo Ear Imaging Preparation. After anesthesia with an i.p. in-jection of ketamine-xylazine, the ear was glued to a glass andplaced on a custom-designed mount.
Intravenous Administration. All dyes were injected i.v. through thelateral tail vein or the retro-orbital venous sinus. An i.v. catheterwas used when timely injection was necessary. For the plasmamarker, 100 μL of 2% wt/wt dextran-conjugated dye was ad-ministered as indicated. To determine the dose of dye for i.v.injection, we multiplied the dilution factor for plasma by theconventional dose of the dye used for tissue staining and modi-fied it experimentally. The following dyes were used: OGB-1:00AM (Invitrogen; 50 μL of 100 μg), Hoechst (20 mg/kg), SR101(Invitrogen; 10 μL of 0.5 mg/mL), adenovirus (100 μL of 1 × 109
pfu of adenoviral vector with CMV promoter-driven GFP ex-pression), Quantum Dot (Q21021MP, Molecular Probes; 20 μLof 2 μM), TAMRA-MION (50 μL of 10 μM), and siRNA (60 μLof 125 pmol mixed with 60 μL of 2% in vivo jetPEI; Polyplus).
1. Isaka N, Padera TP, Hagendoorn J, Fukumura D, Jain RK (2004) Peritumor lymphaticsinduced by vascular endothelial growth factor-C exhibit abnormal function. Cancer Res64:4400–4404.
Choi et al. www.pnas.org/cgi/content/short/1018790108 1 of 6
A 2 sec 10 sec 1 minBaseline
10 sec 1 min 2 minBaseline
Arte
ryC
apilla
ry
Baseline 20 sec 40 sec
60 sec 120 sec 180 sec
C D
B
0 40 80 120 160 20060
80
100
120
Time (s)Fl
uore
scen
ce (r
. f. u
.)
Fig. S1. (A) Laser-induced extravasation in an artery. Arterial stimulation accompanied arterial contraction. The dashed line demarcates the baseline lumenarea. (Scale bar: 50 μm.) (B) Laser-induced extravasation in a capillary. Capillaries were vulnerable to hemorrhage when irradiated. Note the loss of dark streaks,indicating red blood cell movement; a clot formed around the irradiated region (red arrow). (Scale bar: 10 μm.) (C) Transient clot formation after laser ir-radiation. During laser-induced vascular permeabilization, transient clot formation (white arrow) was observed with disturbed downstream blood flow. Thered dot indicates the position of laser irradiation. (Scale bar: 20 μm.) (D) Temporal dynamics of fluorescence intensity in the boxed region shown in C. The arrowindicates the time of laser irradiation.
A B
0 1 2 3 4 5
0
20
40
60
80
100
Energy (mJ)
Succ
essf
ul e
xtra
vasa
tion
(%)
0.5 1.0 2.0 3.0 4.0 5.0
0
10
20
30
40
50Transient clot formationStable clot formation
Energy (mJ)
Res
pons
e (%
)
Fig. S2. (A) Relationship between laser energy and the probability of successful extravasation (n = 18 for each dose of laser energy in three mice). (B) De-pendence of clot formation on laser energy (n = 18 for each dose of laser energy in three mice).
Choi et al. www.pnas.org/cgi/content/short/1018790108 2 of 6
A B
200
μm
Baseline After irradiation
350
μm
Baseline After irradiation ExtravasationC
Fig. S3. Feasibility of laser-induced extravasation in the deep cortical layer and to a large area. (A) 3D view of vascular structure in the cortex. The skull anddura layer were removed, 2 MDa of FITC-dextran was i.v. injected, and images were obtained at 3-μm intervals to a depth of 450 μm. (B) Laser-induced ex-travasation in the deep cortical layers. (Scale bar: 20 μm.) (C) Molecular delivery to a large area by irradiation of multiple blood vessels. Here 70 kDa of FITC-dextran was injected i.v. in the thinned-skull window model, and three extravasations were induced by irradiating three regions in the pial venules. (Scale bar:100 μm.)
A
C 5 sec 20 secBaseline 1 min
B 5 sec 20 secBaseline 1 min
Fig. S4. Red blood cells (RBCs) did not leak during laser-induced vascular permeabilization. RBCs were stained and injected i.v. with TRITC-dextran. (A) StainedRBCs (green) in TRITC-dextran (red) solution. (Scale bar: 10 μm.) (B) RBCs did not leak during laser-induced vascular permeabilization. Green streaks representmovement of stained RBCs. (Scale bar: 20 μm.) (C) RBC leakage during severe arterial contraction by laser irradiation. Severe contraction was induced by RBCleakage after laser irradiation to the artery, as denoted by the clear circular green signals outside of the lumen. (Scale bar: 20 μm.)
Choi et al. www.pnas.org/cgi/content/short/1018790108 3 of 6
A B
D
C
F
0 10 20 30 40 500.0
0.2
0.4
0.6
0.8
1.0
Lumen diameter (μm)
Circ
ular
ity
E
0 10000 20000 30000 40000 500000.0
0.2
0.4
0.6
0.8
1.0
Extravasation area (μm2)
Circ
ular
ity
0 1 2 3 40.0
0.2
0.4
0.6
0.8
1.0
Blood flow (mm/s)
Circ
ular
ity
0 10 20 30 40 500
1
2
3
4
Lumen diameter (μm)
Bloo
d flo
w (m
m/s
)
0 1 2 3 4 50
10000
20000
30000
40000
50000
Blood flow (mm/s)
Extr
avas
atio
n ar
ea (μ
m2 )
Extr
avas
atio
n ar
ea (μ
m2 )
Small venule Large venule0
10000
20000
30000
40000
50000
60000 p = 0.0003
Fig. S5. Characterization of laser-induced extravasation. Lumen diameter and blood flow were measured at baseline, and the extravasation area and cir-cularity of the extravasation area were quantified based on an image obtained at 1 min. (A) Lumen diameter and blood flow in venules showed a strongpositive correlation (R2 = 0.5612; P < 0.0001; n = 37). (B) Compared with large venules (lumen diameter <25 μm; n = 28), small venules (lumen diameter >25 μm;n = 9) showed significantly less extravasation area (P = 0.0003). (C) Blood flow had a nonsignificant negative correlation with extravasation area (R2 = 0.0718;P = 0.1089) (D and E) Smaller venules or venules with slower blood flow had more circular extravasation (R2 = 0.4721; P < 0.0001 vs. R2 = 0.1988; P = 0.0057). (F)The extravasation area was positively correlated with the circularity of extravasation (R2 = 0.2232; P = 0.0032).
Choi et al. www.pnas.org/cgi/content/short/1018790108 4 of 6
Intact skull Thin skull Thin skull + Extravasation
GFA
PIb
a-1
A
B
Open skull
Thin skull Thin skull + Extravasation
Fig. S6. Assessment of delayed cell death and immune response at 2 d after laser-induced extravasation. (A) Nissl staining. Thinned-skull windows (∼1 mm indiameter) were made in each hemisphere, and extravasation was induced in the right hemisphere. (Insets) Magnified views of the thinned-skull windowsregions. (Scale bar: 500 μm.) (B) Immunostaining of astrocyte (GFAP) and microglial (Iba-1) activation. (Scale bar: 100 μm.)
Baseline During extravasation 10 min after extravasation
20 s
10 %∆F/F
Fig. S7. Functional calcium activity of astrocytes during laser-induced plasma leakage. A calcium indicator (OGB-1:00 AM) and astrocyte marker (SR101) wereloaded in the cortex using a conventional bath-loading method. Rhodamine was administered i.v. as a plasma indicator. Astrocyte calcium activity wasmeasured with a 2-Hz acquisition rate. Spontaneous calcium activity in astrocytes is shown.
Choi et al. www.pnas.org/cgi/content/short/1018790108 5 of 6
7 sec 30 sec 1 minBaseline
Qdo
t
B
A
3 sec 8 sec 30 secBaseline
MIO
N
Fig. S8. Laser-induced permeabilization of nanoparticles. (A) Quantum dot. (B) TAMRA- conjugated MiONs.
Head frameThinned Skull
Dental cement
CorticalVasculature
aCSF
20X 1.0 NA
Head frameXYtranslation
stage
A BThinned skull surgery
Intravenous injection of probes
Localization of the target venule
Laser irradiation to the vascular wall
Imaging
Objective
Fig. S9. Experimental setup. (A) Procedure for the laser-induced extravasation experiments. (B) Schematic diagram of the experimental setup.
Choi et al. www.pnas.org/cgi/content/short/1018790108 6 of 6