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https://doi.org/10.1038/s41467-020-14786-z

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Visualization and molecular characterizationof whole-brain vascular networks withcapillary resolution
1234567890():,;

Takeyuki Miyawaki 1 ✉, Shota Morikawa1, Etsuo A. Susaki2,3, Ai Nakashima1, Haruki Takeuchi1,4,
Shun Yamaguchi5,6, Hiroki R. Ueda2,3,7 & Yuji Ikegaya 1,8

Structural elucidation and molecular scrutiny of cerebral vasculature is crucial for understanding the functions and diseases of the brain. Here, we introduce SeeNet, a method fornear-complete three-dimensional visualization of cerebral vascular networks with high signalto-noise ratios compatible with molecular phenotyping. SeeNet employs perfusion of amultifunctional crosslinker, vascular casting by temperature-controlled polymerization of
hybrid hydrogels, and a bile salt-based tissue-clearing technique optimized for observation ofvascular connectivity. SeeNet is capable of whole-brain visualization of molecularly characterized cerebral vasculatures at the single-microvessel level. Moreover, SeeNet reveals ahitherto unidentiﬁed vascular pathway bridging cerebral and hippocampal vessels, thus
serving as a potential tool to evaluate the connectivity of cerebral vasculature.

1 Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan. 2 Department of Systems Pharmacology, Graduate School of Medicine,
The University of Tokyo, Tokyo, Japan. 3 Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, Osaka, Japan. 4 Social
Cooperation Program of Evolutional Chemical Safety Assessment System, LECSAS, Graduate School of Pharmaceutical Sciences, The University of Tokyo,
Tokyo, Japan. 5 Department of Morphological Neuroscience, Graduate School of Medicine, Gifu University, Gifu, Japan. 6 Center for Highly Advanced
Integration of Nano and Life Sciences, Gifu University, Gifu, Japan. 7 International Research Center for Neurointelligence (WPI-IRCN), UTIAS, The Universityof Tokyo, Tokyo, Japan. 8 Center for Information and Neural Networks, National Institute of Information and Communications Technology, Osaka, Japan.
✉email: takeyuki.miyawaki@gmail.com

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Cerebral vasculature is an anatomically and histologicallyinhomogeneous three-dimensional (3D) trafﬁc network
for blood cells, nutrients, oxygen, metabolic wastes, signaling molecules, etc.1–3. Although structural and molecularanalyses of cerebral vasculature are promising for providing
fundamental insights into cerebral circulation and cerebrovascular diseases, which are major causes of death and disability4, few studies have captured detailed structural informationof molecularly identiﬁed vascular networks due to technicallimitations (Supplementary Tables 1 and 2)5–21.
Here, we introduce SeeNet, a method for 3D visualization andmolecular characterization of the entire vascular network,
including virtually all microvessels, in the whole brain. SeeNet
includes (i) intravascular perfusion of a blood–brain barrier(BBB)-impermeable ﬂuorescent crosslinker, (ii) thermally controlled polymerization of a hybrid hydrogel, and (iii) bile saltbased tissue clearing.
Results
Thermally controlled vascular casting with hybrid hydrogel.
Classic methods for vascular casting using solidifying polymers, such as resin11, fail to allow visualization of all vessels
because the polymers often become clogged in microvessels. To
suppress clogging, we conceived a stratagem in which polymerization was initiated after complete perfusion of cerebral
vessels with monomer acrylamide and a BBB-impermeable
ﬂuorescent crosslinker. We designed a ﬂuorescent compound
termed RITC–Dex–GMA consisting of rhodamine isothiocyanate (RITC), a ﬂuorescent group; glycidyl methacrylate(GMA), a crosslinkable group; and dextran (Dex), a macromolecule that makes the compound have a molecular weightlarge enough to prevent it from crossing the BBB and smallenough to not clog microvessels (Fig. 1a). RITC–Dex–GMA
can be synthetized through a few simple chemical reactions
and can easily be puriﬁed with ethanol precipitation (Supplementary Fig. 1a, b)22,23. To regulate the timing of the polymerization of RITC–Dex–GMA and acrylamide, we adopted
the thermal initiator VA-044; note that VA-044 triggers
polymerization when heated to 37 °C (Supplementary Fig. 1c,d). After intravascular perfusion with phosphate-buffered saline (PBS) and paraformaldehyde (PFA), mice were further
perfused with a mixture of RITC–Dex–GMA, acrylamide, andVA-044 at 4 °C. When the sample was maintained at 37 °C for
3 h, a hybrid hydrogel was formed; ﬂuorescent hydrogels
formed inside the vascular lumen, whereas nonﬂuorescent,protein-crosslinked hydrogels formed outside the lumen(Fig. 1b).
We compared the visualization performance of this method(Fig. 1c) to that of intravenous injection or transcardial perfusionof Texas Red-conjugated lectin, which are commonly usedtechniques for vascular staining (Supplementary Figs. 2a and3a). The brain was thin-sectioned and immunolabeled against
CD31, a marker for endothelial cells (Supplementary Figs. 2b and3b). The signal-to-noise (SN) ratio of the vasculature casted by
RITC–Dex–GMA was signiﬁcantly higher than that of thevasculature stained by intravenous injection or transcardial
perfusion of Texas Red-conjugated lectin (Fig. 1d, n = 9, 10,
and 6 slices from 9, 10, and 6 mice, respectively, Dex–GMA
versus lectin (i.v.): P = 1.59 × 10−10, Q2,22 = 16.7; Dex–GMA
versus lectin (perfused): P = 2.01 × 10−5, Q2,22 = 8.27; P = 2.94 ×
10−10, F2,22 = 69.9, Tukey’s test after one-way ANOVA). The SN
ratio of transcardially perfused lectin was signiﬁcantly higher thanthat of intravenously injected lectin (Fig. 1d, n = 6 and 10 slices
from 6 and 10 mice, respectively, P = 4.53 × 10−4, Q2,22 = 6.42,
Tukey’s test after one-way ANOVA). However, as previously2

reported in the liver15, vasculature staining with transcardial
perfusion of lection was spotty (Supplementary Fig. 3). The
overlap ratio of RITC–Dex–GMA-casted vessels and CD31immunopositive vessels was 98.2 ± 0.8%, which was signiﬁcantly
higher than the overlap ratio 59.4 ± 9.45% (intravenous injection)and 64.4 ± 7.32% (perfused) for Texas Red-conjugated lectin(Fig. 1e, mean ± SD of 9, 10, and 6 slices from 9, 10, and 6 mice,
respectively, Dex–GMA versus lectin (i.v.): P = 2.39 × 10−10,
Q2,22 = 16.4; Dex–GMA versus lectin (perfused): P = 3.51 × 10−8,
Q2,22 = 12.4; lectin (i.v.) versus lectin (perfused): P = 0.401, Q2,22= 1.86, P = 1.59 × 10−10, F2,22 = 74.5, Tukey’s test after one-way
ANOVA). Therefore, thermally controlled post hoc polymerization of perfused RITC–Dex–GMA could be used to visualize
nearly all of the cerebral vasculature with a high SN ratio.
We also compared the staining performance between our
method and a gelatin–ﬂuorescein isothiocyanate (FITC)–albumin
gel perfusion method10, which is reported to be able tolabel cerebral vasculature with a high SN ratio (Supplementary
Fig. 4a). Unexpectedly, however, we noticed that thegelatin–FITC–albumin gel perfused samples were only partiallystained with anti-CD31 (Supplementary Fig. 4b–d). The percentage of CD31-positive areas in slices was 4.37 ± 0.94%, in contrast
to the expected 9.25 ± 1.12% when CD31 is successfully stained(Supplementary Fig. 4e, mean ± SD of 5 and 9 slices from 5 and 9
mice, respectively, P = 5.83 × 10−6, t12 = 7.66, Student’s t-test). A
possible explanation for this incomplete staining could be that thewarm PFA used in this method altered the efﬁciency of ﬁxation or
the antigenicity of CD31. To examine whether the antigenicity
was affected, we compared the staining patterns of other protein
markers for neurons (NeuN), astrocytes (GFAP), and microglia(Iba-1) in samples that were ﬁxed using a standard method(PFA), our method (Dex–GMA), or the gelatin–FITC–albumin
method (Supplementary Fig. 5a–c). The immunostaining patterns
in Dex–GMA-treated samples were similar to those of PFA-ﬁxed
samples. However, in gelatin–FITC–albumin-treated samples, theimmunosignals were deteriorated, and the staining patterns
were not spatially even. The percentages of immunopositive
areas to entire areas were similar between PFA-treated and
Dex–GMA-treated samples, whereas the percentages were lower
in gelatin albumin-treated samples (Supplementary Fig. 5d; P =3.47 × 10−3, F2,12 = 9.44, one-way ANOVA; PFA versus
Dex–GMA
versusDex–GMA:P = 0.46,
Q2,12 = 1.73;
gelatin–albumin: ***P = 3.13 × 10−3, Q2,12 = 5.97; PFA versus
gelatin–albumin: *P = 2.79 × 10−2, Q2,12 = 4.238, Tukey’s test.
H2,12 = 9.38,
SupplementaryFig.
5e;
P = 9.19 × 10−3,
Kruskal–Wallis test; PFA versus Dex–GMA: P = 0.10, Q2,12 =
0.99; Dex–GMA versus gelatin–albumin: *P = 2.45 × 10−2,
Q2,12 = 2.61; PFA versus gelatin–albumin: *P = 2.45 × 10−2,
Q2,12 = 2.61, Steel-Dwass test. Supplementary Fig. 5f; P = 7.60 ×
10−4, F2,12 = 13.9, one-way ANOVA; PFA versus Dex-GMA: P =8.48 × 10−2, Q2,12 = 3.342; Dex–GMA versus gelatin–albumin:***P = 5.48 × 10−4, Q2,12 = 7.43; PFA versus gelatin–albumin:**P = 3.35 × 10−2, Q2,12 = 4.09, Tukey’s test.). These data indicatethat the antigenicity was preserved in Dex–GMA-treated andPFA-ﬁxed samples but was altered in gelatin–FITC–albumintreated samples. In the gelatin–FITC–albumin gel perfusion
method, we occasionally observed clusters of CD31-positive
microvessels that were not stained by FITC, suggesting thatgelatin–FITC–albumin gels were clogged in some brain regions(Supplementary Fig. 4f). To quantitatively compare the staining
performance between the two methods, we labeled endothelial
cells using the intravenously injected lectin-conjugated dye for areference (Supplementary Fig. 6a, b). Unlike anti-CD31 staining,
the performances of lectin labeling were similar between the two
methods, possibly because the labeling was carried out before theperfusion of warmed PFA and the change in the antigenicity

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a

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4 °C

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Fig. 1 Near-complete visualization of cerebral vasculature using thermally controlled polymerization of a BBB-impermeable ﬂuorescent crosslinker. a
Design of the BBB impermeable ﬂuorescent crosslinker RITC–Dex–GMA. The ﬂuorophore RITC and the crosslinkable group GMA are chemically linked
through dextran, a BBB-impermeable molecule. b Strategic concept of vascular casting using a hybrid hydrogel. Red circles and gray dots indicate
RITC–Dex–GMA and acrylamide, respectively. c Top: A confocal image of a sagittal section of an adult mouse brain treated with vascular casting using
RITC–Dex–GMA, acrylamide, and VA-044. Scale bar = 1 mm. Bottom: Magniﬁed images of RITC–Dex–GMA-casted vasculature of different brain regions(red) and endothelial cells counterstained using anti-CD31 in the same ﬁelds (green). Scale bar = 100 μm. d SN ratios of the ﬂuorescence intensities in
lectin-based and Dex–GMA-based visualization (n = 10, 6, and 9 slices from 10, 6, and 9 mice, respectively; box plots indicate median and 25–75%
interquartile ranges; whiskers cover 10−90% quantiles. P = 2.94 × 10−10, F2,22 = 69.9, one-way ANOVA; Dex-GMA versus lectin (i.v.); ***P = 1.59 × 10−10,
Q2,22 = 16.7; Dex–GMA versus lectin (perfused); ***P = 2.01 × 10−5, Q2,22 = 8.27; lectin (i.v.) versus lectin (perfused): ***P = 4.53 × 10−4, Q2,22 = 6.42,
Tukey’s test). e Overlap ratios between anti-CD31 immunoreactivity and Texas Red/RITC ﬂuorescence in lectin-based and Dex–GMA-based visualization(59.4 ± 9.45%, 64.4 ± 7.32%, and 98.2 ± 0.8%, mean ± SD of 10, 6, and 9 slices from 10, 6, and 9 mice, respectively; box plots indicate median and 25−75% interquartile ranges; whiskers cover 10−90% quantiles. P = 1.59 × 10−10, F2,22 = 74.5, one-way ANOVA; Dex–GMA versus lectin (i.v.); ***P =2.39 × 10−10, Q2,22 = 16.4; Dex–GMA versus lectin (perfused); ***P = 3.51 × 10−8, Q2,22 = 12.4; lectin (i.v.) versus lectin (perfused): P = 0.401, Q2,22 = 1.86,
Tukey’s test). Source data are provided as a Source Data ﬁle.

(Supplementary Fig. 6c, 10.9 ± 0.41% vs. 10.9 ± 0.54%, mean ±SD of ﬁve slices from ﬁve mice each, P = 0.892, t8 = 0.14,
Student’s t-test). Again, the clusters of lectin-positive vesselsthat were not stained by FITC were occasionally observed ingelatin–FITC-treated samples (Supplementary Fig. 6a), and theperformance of vasculature staining was higher in the
FITC–Dex–GMA-based protocol (Supplementary Fig. 6d,
95.9 ± 1.97% vs. 98.3 ± 0.49%, mean ± SD of ﬁve slices fromﬁve mice each, P = 4.72 × 10−2, t8 = 2.34, Student’s t-test).
Taken together, we consider that our method is more suitable

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Days
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Fig. 2 Rapid tissue clearing with sodium deoxycholate. a, b Time-series of photographs of a brain taken at each step of tissue clearing. The ﬁxed brain was
treated with EDTA for 12 h followed by SDS a or SDC b for 7 d (daily refreshed), washed with PBS and refractive index (RI)-matched in ScaleCUBIC-2 for 2
d. In the SDS-treated group, the white matter remained opaque, and the sample swelled. In the SDC-treated group, the cleared brain was still pale yellow,
but the white matter was transparent. The sample swelled to a lesser extent. The grid interval is 2 mm. c The light transmittance (380–780 nm) of ﬁxed
whole brains treated with SDS and ScaleCUBIC-2 (dark blue), with SDC and ScaleCUBIC-2 (light blue) or with PBS (black) (n = 8 brains each,P = 2.22 × 10−16, F2,889 = 4.56 × 104, two-way ANOVA (between groups)). d Percentages of opaque area in the whole brains treated with SDS andScaleCUBIC-2, with SDC and ScaleCUBIC-2, or with PBS (n = 8 brains each; box plots indicate median and 25−75% interquartile ranges, P = 2.22 × 10−16,
F2,21 = 4.08 × 103, one-way ANOVA; SDC versus SDS: ***P = 7.47 × 10−14, Q2,21 = 26.73; SDS versus PBS: ***P = 4.36 × 10−14, Q2,21 = 94.78; SDC versus
PBS: ***P = 4.36 × 10−14, Q2,21 = 121.5, Tukey’s test). e Time course of tissue expansion during clearing (n = 8 brains each, P = 2.22 × 10−16, F2,21 = 6.66 ×
102, one-way ANOVA; SDC versus SDS: ***P = 4.41 × 10−14, Q2,21 = 33.15; SDC versus PBS: ***P = 9.92 × 10−11, Q2,21 = 17.69; SDS versus PBS: ***P =4.36 × 10−14, Q2,21 = 50.84, Tukey’s test). Error bars represent standard deviations. Source data are provided as a Source Data ﬁle.

for analyzing the molecular and structural characteristics of
cerebral vasculature.
Vascular connectivity-retaining delipidation by a bile salt. For
the visualization of the 3D structure of vascular networks, conventional casting methods use corrosive chemicals and remove
surrounding parenchymal tissues. We sought to replace thisclassical strategy with optical tissue clearing that retains
4molecular information in the brain parenchyma. First, we conducted tissue clearing by delipidation with sodium dodecyl sulfate(SDS) for 1 week5,13 followed by refractive index (RI) matchingwith ScaleCUBIC-2, an aqueous solution of sucrose, urea, andtriethanolamine, for 2 d24. However, SDS-based delipidation
insufﬁciently cleared the white matter of the brain and caused thespecimens to become fragile and swell (Fig. 2a). We thus usedsodium deoxycholate (SDC), a bile salt that has been used forclearing of plant tissues25. Although SDC has been considered

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SDS

b

c
Torn penetrating vessel (%)

a

SDC

***
100

75

50

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C
SD

SD

S

0

Fig. 3 Breakage of the casted hydrogel is suppressed in SDC-based tissue clearing. a, b Maximum projection of volumetric images of the neocortex. The
vascular-casted samples were delipidated using SDS a or SDC b and were immersed in ScaleCUBIC-2. Some penetrating vessels were severed(arrowheads) in the SDS-treated group, whereas such breakages were rarely observed in the SDC-treated group. Scale bar = 100 μm. c The percentages ofthe broken penetrating vessels in the brain treated with SDS and SDC (n = 6 and 9 brains, respectively; box plots indicate median and 25−75%
interquartile ranges, ***P = 8.84 × 10−7, t13 = 8.70, Student’s t-test). Source data are provided as a Source Data ﬁle.

inapplicable for use in animal tissues13,24, we found that it madethe mouse brain transparent after 2-d RI matching with ScaleCUBIC-2, at least under our ﬁxation condition (Fig. 2b). The
average light-transmittance ratio of SDC-cleared tissues was
slightly lower than that of SDS-cleared tissues (Fig. 2c, n = 8
brains each, P = 2.22 × 10−16, F2,889 = 4.56 × 104, two-way
ANOVA), but SDC-cleared tissues exhibited a spatially uniform
RI across the specimens and had no opaque areas (Fig. 2d, n = 8
brains each, P = 7.47 × 10−14, Q2,21 = 26.73; SDC versus SDS,
P = 2.22 × 10−16, F2,21 = 4.08 × 103, Tukey’s test after one-way
ANOVA). Moreover, swelling was signiﬁcantly reduced in
SDC-cleared tissues (Fig. 2e, n = 8 brains each, P = 4.41 × 10−14,
Q2,21 = 33.15 versus the SDS group, P = 2.22 × 10−16, F2,21 = 666,
Tukey’s test after one-way ANOVA). Thus, the connectivity ofthe vascular network was preserved in SDC-cleared tissues,
whereas many neocortical penetrating vessels were fractured in
SDS-cleared tissues due to overstretching of the ﬂuorescenthydrogel (Fig. 3, n = 6 and 9 brains. P = 8.84 × 10−7, t13 = 8.70,
Student’s t-test). We refer herein to the combination of ﬂuorescent Dex–GMA-based vascular casting and SDC-based tissue
clearing as SeeNet (A method to See the cerebrovascular
Network).
Molecular phenotyping compatibility of SeeNet. Next, we
examined whether nucleic acids and proteins were maintained in
SeeNet-treated brains. The vasculatures were cast using
FITC–Dex–GMA, and the specimens were labeled using propidium iodide (PI), a DNA-staining reagent, or an antibody against
Iba-1. Whole-mount imaging of the cerebral hemisphere revealed
that PI and Iba-1 signals were detectable (Fig. 4a–f, n = 7
hemispheres each), suggesting that nucleic acids and proteins
were retained in the tissues.
We also addressed the possible quenching of ﬂuorescentproteins during the clearing procedure. We applied SeeNet to thebrains of visually stimulated Arc-dVenus mice, in which active
neurons express the modiﬁed yellow ﬂuorescent protein dVenus26. Whole-mount imaging revealed that SeeNet retained theﬂuorescence of dVenus (Fig. 4g–i, n = 7 hemispheres).
We further examined the compatibility of SeeNet with other
epitopes and ﬂuorescent proteins using a light-sheet ﬂuorescencemicroscope7. First, SeeNet-treated hemispheres were immunostained with ﬂuorophore-conjugated antibodies against NeuN and
GFAP (Supplementary Fig. 7). The optical sections of the stained
samples were virtually indistinguishable from images of 2D-

stained samples (Supplementary Figs. 5a, b and 7c, f), indicatingthat SeeNet-treated samples can be immunostained using these
antibodies. Next, SeeNet protocol was applied to CX3CR1-GFP
mice27, which expresses GFP in microglia, and H-I7-iCreimCherry mice28, which expresses mCherry in a subpopulationof olfactory sensory neurons (Supplementary Fig. 8a–f). In
CX3CR1-GFP mice, GFP-positive cells were observed throughout
the brain. In H-I7-iCre-imCherry mice, the convergence patternof mCherry-positive axons were clearly visualized with SeeNet
protocol. These patterns were consistent with the expression
patterns reported previously in these mouse lines, suggesting that
the SeeNet protocol preserved the innate ﬂuorescence of GFP and
mCherry. In addition, SeeNet was able to visualize ﬂuorescentprotein expressed by intracranially injected adeno-associatedvirus (AAV), without apparent leakages of FITC–Dex–GMA
around the injected sites (Supplementary Fig. 8g–i).
We next examined whether the ﬁne subcellular structuresof glial cells were preserved in the SeeNet protocol. GFAP-stained
or CX3CR1-GFP-expressing SeeNet-treated brains were imaged
by a confocal microscope. We successfully observed typicalglial subcellular morphologies, i.e., astrocytic endfeet wrapping
penetrating vessels29 and microglial protrusions that likely
touched capillaries30 (Supplementary Fig. 9).
Then, we compared the SN ratio of 3D imaging data obtained
from the SeeNet-treated samples to that obtained from samples
prepared by a conventional protocol, intravenous injection of
Texas Red-conjugated lectin and optical clearing with SDS/
ScaleCUBIC-2 (Supplementary Fig. 10). Signals in SeeNet-treatedbrains were detectable at depths of at least 1000 μm, whereas
signals in lectin/SDS/ScaleCUBIC-2-treated brains were almost
indistinguishable from the background (Supplementary Fig. 10a,b, n = 7, 9 mice, respectively, P = 2.22 × 10−16, F1,140 = 5.55 ×
102; two-way ANOVA). The mean diameter of cerebral capillaries
in SeeNet-treated samples was 6.58 ± 1.21 µm (Supplementary
Fig. 10c, mean ± SD of 90 capillaries from 9 mice). This value wasslightly but not signiﬁcantly larger than the previously reported
in vivo diameter (6.3 ± 1.1 µm)31, possibly due to changes intissue volume caused by PFA ﬁxation, delipidation, and RImatching.
As the capillary diameters in SeeNet-treated samples werecomparable to the pixel resolution of our macro-zoom light-sheet
microscope, in which one pixel measures 6.45 × 6.45 µm2, we
attempted whole-brain imaging of cerebral vasculature at singlecapillary resolution. Because of the high SN ratios of SeeNettreated brain, we were able to visualize blood vessels in both gray

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FITC-Dex-GMAPIb

Z = 200 µmbFITC-Dex-GMAe

Z = 200 µmey = –0.0002x + 0.986 (n = 7)
R 2 = 0.0072

0.5

0.0
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Distance from vessel (µm)

f1.0
y = –0.0007x + 1.00 (n = 7)
R 2 = 0.282

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Arc-dVenus (visual stim.)h

Z = 600 µm @ V1hi
Fluorescence (Normalized)

RITC-Dex-GMA

g

1.0

Iba1

Fluorescence (Normalized)dcFluorescence (Normalized)a1.0
y = 0.0002x + 1.00 (n = 7)
R 2 = 0.343
0.5

0.0
0
10 20 30 40
Distance from vessel (µm)

Fig. 4 SeeNet retains nucleic acids, proteins, and ﬂuorescence of endogenous probes. a Maximum projection of volumetric images of the cerebral
hemisphere. The vasculature was cast using FITC–Dex–GMA (green). After delipidation with SDC, nucleic acids were labeled using PI (magenta). Then, thesample was RI-matched using ScaleCUBIC-2. Scale bar = 1 mm. b An optical section of a boxed region in panel a at Z = 200 μm. PI ﬂuorescence was
uniformly retained across the section. Scale bar = 100 μm. c The normalized median intensities of PI ﬂuorescence were plotted as a function of the distancefrom the nearest vessel. The intensities were almost unchanged depending on the distance. n = 7 hemispheres from seven mice. d–f The same as panels
a–c showing the immunohistochemical signal of anti-Iba-1 (magenta). g–i The same as panels a–c showing RITC–Dex–GMA (magenta) and Arc-dVenus(green). Error bars represent standard deviations. Source data are provided as a Source Data ﬁle.

and white matters of the brains with single-capillary resolution(Fig. 5a–d, Supplementary Movies 1 and 2). The brains were also
immunostained using a FITC-conjugated antibody against αsmooth muscle actin (αSMA), a marker for arteries and arterioles.
SeeNet could visualize the whole cerebral vasculature withmolecularly identiﬁed arterioles and venules (Fig. 5a).
We also examined the SN ratio of 3D imaging data obtained
from samples prepared with intravenous injection of an Alexa
647-conjugated anti-CD31 antibody and with optical clearing by
ethanol and ethyl cinnamate (ECi)32. One sample was preparedfrom CX3CR1-GFP mice for the simultaneous examination of theﬂuorescence preservation of this method (Supplementary
Fig. 11a–c). GFP ﬂuorescence was barely visible in the graymatter of the brains and was almost invisible in the white matter(Supplementary Fig. 11d–f). The signal of endothelial cells was
detectable in the gray matter but indistinguishable from thebackground noise in the white matter (Supplementary Fig. 11g–i).
The SN ratio of the CD31 signal was signiﬁcantly lower than that
6of casted vessels in SeeNet (Supplementary Fig. 11j, k. n = 4 and 5
optical slices from 4 or 5 mice, respectively. Gray matter; ***P =1.32 × 10−6, t7 = 15.1, White matter; ***P = 2.53 × 10−5, t7 =9.75, Student’s t-test).
A microvascular path revealed by SeeNet. Using SeeNet, we
serendipitously discovered a previously unknown microvascularpath. In rodents, the vascular paths of the hippocampus and the
cerebral cortex were examined at the level of arterioles andvenules. Once cerebral arteries are separated into the posterior
cerebral artery and the longitudinal hippocampal artery, they are
generally assumed to form independent networks until they
converge in the internal jugular vein1,33. However, when we
inspected SeeNet-treated samples at the single-capillary level, we
found some microvessels that directly connected cortical andhippocampal vascular networks (Fig. 5e, Supplementary
Movies 3, 4). We analyzed 54 arterioles and 57 venules of the

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a

RITC-Dex-GMAb

Z = 3 mm

αSMA-FITCcdc

αSMA-FITCRITC-Dex-GMA

e

d

g

f1

Penetrating arteriole
Penetrating venuleNot connected

100

f2

HPC

CTX

f2

Connected penetrating
vessel (%)

f3

f3

f1
CTX
CTX
HPC

50
25
0

HPC

Hipocampal
arteriole

Hippocampal
venuleαSMA-FITC (Confocal)

RITC-Dex-GMA (Confocal)ij

Penetratingarteriole

h

75

CTX

Capillary

HPC

al
mp
oca
p
Hip rioleeartFig. 5 Large-scale molecular phenotyping-compatible 3D imaging of cerebral vasculature. a Maximum projection of light-sheet ﬂuorescence microscopic
images of the entire vasculature in a SeeNet-treated brain (1.6× zoom, ×0.63 objective lens, numerical aperture: 0.15, working distance: 87 mm) into asingle stacked photo. Casted vessels are shown in red. Arterioles immunolabeled with anti-αSMA are shown in green. Scale bar = 1 mm. b The
RITC–Dex–GMA signal in a, shown on a grayscale, was optically sectioned at Z = 3 mm. Scale bar = 1 mm. c, d Magniﬁed images of the boxed region ofb. Vasculature in the gray matter c and white matter d was visualized with capillary resolution. Scale bar = 200 μm. e The RITC–Dex–GMA signal in a
posterior part of the left hemisphere in a is shown on a grayscale. Magenta indicates cortico-hippocampal vascular paths observed in this area. Scale bar =1 mm. See also Supplementary Movie 3. f The anti-αSMA immunosignal of the boxed regions in e is shown on a green scale. Magenta contours indicate
cortico-hippocampal vascular paths. Each end of the vascular path is denoted by CTX (cortical side) or HPC (hippocampal side). Arteriole–arteriole (f1),venule–arteriole (f2), and venule–venule (f3) connections were observed. Scale bar = 100 μm. See also Supplementary Movie 4. g The percentage of theconnected pairs of dorsal hippocampal transverse arterioles (red) and venules (blue) to the total number of vessels observed. h Maximum projection ofconfocal images of casted vasculatures from the pia to the hippocampus. A cortico-hippocampal vascular path is shown in magenta. Scale bar = 100 μm.
See also Supplementary Movie 5. i The anti-αSMA immunosignal in the same ﬁeld is shown on a green scale. Scale bar = 100 μm. See also Supplementary
Movie 6. j A 3D-rendered view of the cortico-hippocampal vascular path. Source data are provided as a Source Data ﬁle.
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dorsal hippocampal transverse vessels from three mice (Fig. 5f, g).
We found that 16.7% of the arterioles and 17.5% of the venules
were connected to cortical penetrating vessels, suggesting that theﬂows of blood were not unidirectional. Interestingly, there were
not only arteriole–venule connections (ﬁve hippocampal
arteriole–cortical venule connections and four hippocampal
venule–cortical arteriole connections) but also arteriole–arteriole
and venule–venule connections (four and six connections,
respectively). We conﬁrmed, using higher resolution confocal
imaging, that there is a cortico-hippocampal microvascularpathway connecting αSMA-immunopositive penetrating vessels
and transverse vessels (Fig. 5h–j, Supplementary Movies 5, 6).
These bypasses may actively exchange arterial blood and serve asa backup circulation pathway in the case of cerebral infarction34.
Discussion
In this study, we developed SeeNet, a method for the structuraland molecular phenotyping of the 3D cerebral vasculature. The
BBB-impermeable ﬂuorescent crosslinker RITC–Dex–GMA
enabled near-complete vascular casting with high SN ratios, andSDC-enabled relatively rapid (~10 d) tissue clearing without signiﬁcantly affecting the synthesized ﬂuorescent hydrogels or
intrinsic nucleic acids or proteins.
Several pioneering tissue-clearing studies have attempted
whole-brain visualization of cerebral vasculature using conventional vascular-labeling dyes17,35,36. However, due to the low SN
ratios of the conventional dyes, these methods produce numerous
false negatives. Although Di Giovanna et al. addressed this matter
using temperature-dependent gelation of gelatin and FITCconjugated albumin10,14, the gelatin-based method has anothersource of false negatives because gelatin occasionally becomesclogged in microvessels. Moreover, when samples prepared bythis protocol are molecularly characterized, further caution isnecessary because antigenicity may be altered. In addition, thegelatin–FITC–albumin gel perfusion method costs > $200 per
mouse and takes >6 weeks, making its use for routine vascularlabeling difﬁcult. To reduce false negatives, the use of transgenic
mice is an alternative option. However, because the promoter for
endothelial cells is heterogeneously active, particularly in adultmice37, Cre-based double transgenic mice are required for complete labeling of cerebral vasculatures18,20. Therefore, the supplyof double-transgenic mice is a bottleneck for research, and the useof double transgenic mice makes exploiting other transgenic mice
difﬁcult. In terms of these issues, SeeNet is quick, less costly andeasily adoptable to wild-type and transgenic mice. SeeNet will
enable the routinization of ﬁne large-scale 3D tracing for molecularly characterizable cerebral vasculatures, thus advancing ourunderstanding of the structural and functional organizations of
cerebrovascular networks.
Methods

Animal ethics. Two- to ﬁve-month-old C57BL/6J (Japan SLC, Inc., Japan),
CX3CR1 -GFP (Stock No. 005582, The Jackson Laboratory)27, H-I7-iCreimCherry (Accession No. CDB0537T, RIKEN LARGE)28, and Arc-dVenus transgenic mice26 were used in this study. All animals were housed under a 12:12-h
dark–light cycle (light from 07:00 to 19:00) at 22 ± 1 °C with ad libitum access tofood and water. Animal experiments were performed with the approval of theAnimal Experiment Ethics Committee at the University of Tokyo (approval
number: 24-70) and according to the University of Tokyo guidelines for the Careand Use of Laboratory Animals. All experimental protocols were carried out inaccordance with the Fundamental Guidelines for Proper Conduct of Animal
Experiment and Related Activities in Academic Research Institutions (Ministry of
Education, Culture, Sports, Science and Technology, Notice No. 71 of 2006), theStandards for Breeding and Housing of and Pain Alleviation for Experimental
Animals (Ministry of the Environment, Notice No. 88 of 2006) and the Guidelines
on the Method of Animal Disposal (Prime Minister’s Ofﬁce, Notice No. 40
of 1995).
8Synthesis of RITC–Dex–GMA or FITC–Dex–GMA. The reactions are summarizedin Supplementary Fig. 1. Step 1 (Supplementary Fig. 1a): A total of 4.0 g dextran 40(049-22331, Wako Pure Chemical Industries Ltd., Japan) or dextran 200 (04322611, Wako Pure Chemical Industries Ltd.) was dissolved in 36 ml DMSO
(13406-55, Nacalai Tesque Inc., Japan) in a 50-ml conical tube under a nitrogen
atmosphere. After dissolution of 0.8 g 4-dimethylaminopyridine (12922-02, Nacalai
Tesque Inc.), 2.0 g GMA (17107-55, Nacalai Tesque Inc.) was added. The solution
was gently shaken at 37 °C for 72 h. The reaction was stopped by adding anequimolar amount of concentrated HCl (18320-15, Nacalai Tesque Inc.) to neutralize 4-dimethylaminopyridine. The reaction mixture was precipitated by adding
4 ml of 3 M NaOAc (192-01075, Wako Pure Chemical Industries Ltd.) and 28 ml ofisopropanol (29113-95, Nacalai Tesque Inc.). Then, the resulting ﬂuffy product
Dex–GMA was centrifuged at 6000 × g at 4 °C for 15 min. The precipitates were
further washed in 5 ml of 70% EtOH (14713-95, Nacalai Tesque Inc.) and centrifuged again at 6000 × g at 4 °C for 15 min. Step 2 (Supplementary Fig. 1b): The
resulting Dex–GMA was dissolved in 40 ml of DMSO containing a few drops ofpyridine (162-05313, Wako Pure Chemical Industries Ltd.). Forty milligrams of
RITC (R1755, Sigma-Aldrich, St. Louis, MO, USA) or FITC (7250, Sigma-Aldrich)
was added, followed by the addition of 80 mg of dibutyltin dilaurate (D0303, Tokyo
Chemical Industry Co., LTD., Japan). The mixture was heated at 98 °C for 3 h while
it was stirred. The reaction mixture was cooled at room temperature and precipitated by adding 4 ml of 3 M NaOAc and 28 ml of isopropanol. Then, theresulting ﬂuffy product RITC–Dex–GMA or FITC–Dex–GMA was centrifuged at
6000 × g at 4 °C for 15 min. The precipitate was further washed with 5 ml of 70%
EtOH and centrifuged again at 6000 × g at 4 °C for 15 min. The product was storedat −20 °C. At the time of use, the product was dissolved in ~80 ml of PBS to form a
5% RITC/FITC–Dex–GMA solution.
Vascular casting with RITC–Dex–GMA or FITC–Dex–GMA. A mixture of 5%
RITC–Dex–GMA, 5% acrylamide (011-08015, Wako Pure Chemical Industries
Ltd.), and 0.25% VA-044 (017-19362, Wako Pure Chemical Industries Ltd.) was
prepared in PBS on ice. We usually prepared 10 ml of the mixture containing
dextran 40-based RITC–Dex–GMA and another 10 ml of the mixture containing
dextran 200-based RITC–Dex–GMA. The mixtures were separately ﬁlteredthrough Whatman® qualitative ﬁlter papers (Grade 1, 1001 125, Sigma-Aldrich)that had been premoistened with PBS. Note that elevation in the temperature,
degassing, and lack of ﬁltration may lead to insufﬁcient casting. Then, mice wereanesthetized with 25 mg/kg pentobarbital (SOM04-YO1706, Kyoritsu Seiyaku
Corporation, Japan) and 10 mg/kg xylazine hydrochloride (593-12891, Wako Pure
Chemical Industries Ltd.). Using a peristaltic pump (Minipuls 3, Gilson, Middleton, WI), the mice were perfused with 20 ml of PBS containing heparin (10 U/
ml, 17513-41, Nacalai Tesque Inc.) and verapamil (0.2 mg/ml, 222-00781, Wako
Pure Chemical Industries Ltd.), 20 ml of 4% PFA, 10 ml of 5% RITC–Dex–GMA(dextran 40 based), 5% acrylamide, and 0.25% VA-044 and 10 ml of 5%
RITC–Dex–GMA (dextran 200 based), 5% acrylamide and 0.25% VA-044 at 4 °C
at 4 ml/min in this order. During perfusion, all perfusate-containing equipment
was placed on ice. After the perfusion was complete, the mice were transferred into
50-ml conical tubes with their head down. Then, the tube was degassed to replace
the air with nitrogen because oxygen in the air impedes hydrogel formation. The
tube was submerged in a 37 °C water bath and incubated for exactly 3 h (longer
incubation would make delipidation slower). Vascular casting by FITC–Dex–GMA
was carried out in the same manner.
Vascular labeling with ﬂuorophore-conjugated lectin. Mice were anesthetizedwith 25 mg/kg pentobarbital (SOM04-YO1706, Kyoritsu Seiyaku Corporation) and
10 mg/kg xylazine hydrochloride (593-12891, Wako Pure Chemical Industries
Ltd.). For labeling by intravenous injection, the tail of the mouse was placed in a
beaker ﬁlled with warm water at 40 °C for 30 s so that the tail veins became dilated.
One hundred microliters of Texas Red-conjugated lectin (Texas Red®-labeled
Lycopersicon Esculentum lectin, TL-1176, Vector Laboratories, Burlingame, CA)or DyLight594-conjugated lectin (DyLight® 594 labeled Lycopersicon Esculentum
Lectin, TL-1177, Vector Laboratories) was slowly injected via a lateral tail vein witha 27-G needle. More than 5 min after injection of lectin, animals were perfusedwith 20 ml of heparinized PBS, 20 ml of PFA, and 20 ml of acrylamide and VA044.
For labeling via transcardial perfusion, anesthetized mice were perfused with 20 mlof heparinized PBS, 20 ml of PBS containing 100 μl of Texas Red-conjugated lectin,
20 ml of PFA, and 20 ml of acrylamide and VA-044.
Vascular labeling with gelatin and FITC-albumin. A gel solution containing 2%
porcine skin gelatin type A (G1890, Sigma-Aldrich) was prepared in boiled PBS
and cooled to <50 °C. The solution was then combined with 1% FITC-labeled
albumin (A9771, Sigma-Aldrich), maintained with stirring at 40 °C, and ﬁlteredthrough Whatman® qualitative ﬁlter papers (Grade 1) that had been premoistenedwith PBS. The ﬁltered solution was maintained at 40 °C before the perfusion. Then,
mice were anesthetized with 25 mg/kg pentobarbital and 10 mg/kg xylazinehydrochloride. Using a peristaltic pump, the mice were perfused with 60 ml of PBS
containing heparin (10 U/ml, 17513-41, Nacalai Tesque Inc.) and verapamil (0.2
mg/ml, 222-00781, Wako Pure Chemical Industries Ltd.), and 60 ml of 4% PFA. All
solutions were maintained at 40 °C. After the perfusion of PFA, 20 ml of the

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ﬂuorescent gel perfusate was manually perfused at ~0.6 ml/s, with the body of themouse tilted by 30° and the head down. The body was submerged in ice water torapidly cool and solidify the gel as the ﬁnal portion of the gel perfusate was pushed
through. The brain was carefully extracted after ∼15 min of cooling and submerged
in 4% PFA at 4 °C for 12–16 h.
Clearing protocol. To minimize potential damage to the pial vessels, the brains of
the ﬁxed samples were removed together with their skulls. Then, the brains weretransferred to 15-ml conical tubes containing 10 ml of 0.5 M EDTA (pH 8.0,15130-95, Nacalai Tesque Inc.). The samples were decalciﬁed overnight at 37 °C
while shaking. Then, the softened skulls were gently removed from the shrunken
brains. The brain-hydrogel matrices were transferred into 50-ml conical tubes
containing 20 ml of 10% SDC (194-08311, Wako Pure Chemical Industries Ltd.) or
8% SDS (30400-85, Nacalai Tesque Inc.) in 200 mM boric acid (021-02195, Wako
Pure Chemical Industries Ltd.)-based buffer at pH 8.5. Then, the samples wereincubated for 7–10 d at 37 °C while shaking. The buffers were refreshed daily or
every 2 d. The delipidated samples were washed three times in PBS at room
temperature for 2 h each and were placed in 20 ml of ScaleCUBIC-2 at 37 °C for
2 d. ScaleCUBIC-2 was prepared as a mixture of 50 wt% sucrose (30403-55, Nacalai
Tesque Inc.), 25 wt% urea (35907-15, Nacalai Tesque Inc.), and 10 wt% 2,20,20’nitrilotriethanol (145-05605, Wako Pure Chemical Industries Ltd.).
Vascular visualization with anti-CD31 and EtOH-ECi. Two- to ﬁve-month-old
CX3CR1-GFP or wild-type mice were anesthetized with 25 mg/kg pentobarbital
and 10 mg/kg xylazine hydrochloride. Then, mice were treated with intravenous
injection of 10 µg anti-CD31-Alexa 647 (102416; BioLegend, San Diego, CA, USA)in 100 µl saline solution. After 10 min, the mice were perfused using a peristaltic
pump with 20 ml of PBS containing heparin (10 U/ml) and verapamil (0.2 mg/ml),
and 20 ml of 4% PFA. After perfusion, the brain or the liver was removed and postﬁxed with 4% PFA at 4 °C overnight. The brains were dehydrated in a gradedethanol (14712-05, Nacalai Tesque Inc.) series of 50% (24 h), 70% (24 h), and 100%
(24 h, twice) at 4 °C with gentle shaking. The pH of EtOH was adjusted to 9.0 using
trimethylamine (T0424, Tokyo Chemical Industry Co., LTD.). After dehydration,
the samples were transferred to ECi (BD129295-500g; BLD Pharmatech Ltd.,
Shanghai, China) and incubated with gently shaking at room temperature until
they became transparent (4–5 h).
2D immunostaining. The ﬁxed samples were immersed in PBS containing 15%
sucrose at 4 °C overnight and then in PBS containing 30% sucrose at 4 °C for 2 d.
The samples were immersed in optimum cutting temperature (O.C.T.) compound
and were frozen with dry ice. Sagittal sections at a 40-µm thickness were preparedat −20 °C using a cryostat (HM520, Thermo Fisher Scientiﬁc, Waltham, MA,
USA). Immunohistochemistry was conducted on glass slides. Slices were rinsed in
PBS at room temperature for 15 min and then permeabilized at room temperaturefor 30 min in 0.1 M phosphate buffer and 0.3% Triton X-100 (35501-15, Nacalai
Tesque Inc.) with 10% goat serum (S-1000, Vector Laboratories). The samples were
subsequently incubated with rat anti-CD31 (1:300; 550274, BD Biosciences,Franklin Lakes, NJ, USA), rabbit anti-NeuN (1:300; ABN-78, Sigma-Aldrich),rabbit anti-GFAP (1:300; Z0334, Agilent Technologies, Santa Clara, CA), or rabbitanti-Iba1 (1:300; 019-19741, Wako Pure Chemical Industries Ltd) in 0.1 M phosphate buffer and 0.3% Triton X-100 with 10% goat serum at room temperature
overnight. After the samples were rinsed in 0.1 M phosphate buffer for 3–5 min,they were incubated with the secondary antibody Alexa 488-labeled or Alexa 594labeled goat anti-rat IgG (1:300; A-11006 or A-11007, Thermo Fisher Scientiﬁc) or
Alexa 594-labeled donkey anti-rabbit IgG (1:300; R-37119, Thermo Fisher Scientiﬁc) in 0.1 M phosphate buffer with 10% goat serum at room temperature for 4 h.
After the samples were rinsed with 0.1 M phosphate buffer for 3 × 10 min, they
were mounted using VECTASHIELD Antifade Mounting Medium (H-1000,
Vector Laboratories).
3D immunostaining and nuclear staining. Delipidated samples were immunostained using rabbit anti-Iba1 (1:200; 019-19741, Wako Pure Chemical Industries
Ltd.), Alexa 488-conjugated mouse anti-NeuN (1:100; MAB377X, Merck-Millipore,
Burlington, MA, USA), Cy3-conjugated mouse anti-GFAP (1:100; C9205, SigmaAldrich), or FITC‐conjugated mouse anti-αSMA (1:100; F3777, Sigma-Aldrich) in
0.5% (v/v) Triton X-100, 0.25% casein (12922-02, Nacalai Tesque Inc.), and 0.05%
sodium azide (31208-82, Nacalai Tesque Inc.) at 37 °C for 6 d (anti-Iba1) or 15 d(anti-NeuN, anti-GFAP, anti-αSMA) while shaking. The total volume of thestaining solution was 800 µl for the whole brain and 400 µl for the hemisphere.
Anti-Iba1-treated samples were then washed three times in PBS for 2 h each andtreated with Alexa 594-labeled goat anti-rabbit IgG (1:300; A-11012, Thermo
Fisher Scientiﬁc) at 37 °C for 6 d with shaking. The stained samples were washedthree times in PBS at room temperature for 2 h each and immersed in ScaleCUBIC2. Except for anti-αSMA-stained samples, immunostained samples were reﬁxed
with 1% formaldehyde (16223-55, Nacalai Tesque Inc.) at 25 °C for 24 h before the
RI matching with ScaleCUBIC-2. For nuclear staining, the delipidated samples
were immersed in PBS containing 5 μg/ml PI (P3566, Thermo Fisher Scientiﬁc),
0.5% (v/v) Triton X-100, and 0.05% sodium azide at 37 °C for 12 d with shaking.
The solution was refreshed every 3 d.

ARTICLEAAV production. pAAV-CaMKIIa-mCherry-WPRE-hGH-polyA was constructedfrom pAAV-CaMKIIa-hM3D(Gq)-mCherry-WPRE-hGH-polyA (Addgene 50476)
using SalI (1080 A; TaKaRa Bio Inc., Japan), EcoRV (1042A; TaKaRa Bio Inc.), andthe In-Fusion HD Cloning Kit (Z9648N; TaKaRa Bio Inc.). Recombinant AAV wasgenerated by triple transfection of the 293 AAV cell line (AAV-100; Cell Biolabs,Inc.) with AAVdj rep-cap and pHelper from the AAV-DJ Helper Free Packaging
System (VPK-400-DJ; Cell Biolabs, Inc., San Diego, CA) and pAAV-CaMKIIamCherry-WPRE-hGH-polyA, using PEI-max (24765; Polysciences, Inc., Warrington, PA). AAV vectors were puriﬁed using the AAVpro Puriﬁcation Kit All
Serotypes (6666; Takara Bio Inc.). Virus titers were then determined by qPCR
using the primer pair AAV2 ITR38, Luna Universal® qPCR Master Mix (M3003S;
New England Biolabs, Ipswitch, MA), and the LightCycler® qPCR 2.0 system(DX400; Roche, Basel, Switzerland).
Virus injection. Mice anesthetized with isoﬂurane (Pﬁzer Inc., New York, NY)
were placed in a stereotaxic frame following subcutaneous administration ofropivacaine (100 µl; Aspen Japan, Japan). rAAVdj-CaMKIIa-mCherry (1.5 × 1013
GC/ml, 250 nl) was pressure-injected into the primary somatosensory area (S1;
caudal −1 mm, lateral ± 2.25 mm from the bregma, and ventral 0.5 mm from thesurface of the brain) and the primary visual cortex (V1; caudal −3.5 mm, lateral ±2.5 mm from the bregma, and ventral 0.5 mm from the surface of the brain) at a
rate of 50 nl/min using a syringe pump (KD Scientiﬁc Inc., Holliston, MA, USA)
connected to glass pipettes (30-0034; Harvard Apparatus, Holliston, MA). After
intraperitoneal administration of meloxicam (100 μl of 0.5 mg/ml; Boehringer
Ingelheim Vetmedica Inc., Duluth, GA) at the end of surgery, the mice werereturned to their home cages. SeeNet was conducted 2 weeks after the virus
injection.
Light transmittance measurements. Vascular casting of wild-type mice was
performed with Dex–GMA (nonﬂuorescent), acrylamide, and VA-044. Then, thesamples were decalciﬁed, delipidated, and RI adjusted, as described above. Lighttransmittance was measured from 380 to 780 nm at 5-nm intervals with an integrating sphere (Spectral Haze Meter SH 7000, Nippon Denshoku Industries,
Japan). The samples were placed at the center of the optical cell (2277, Nippon
Denshoku Industries). Two values were measured by the instrument: diffused(scattered) light transmittance (%), which is the value of the light transmitted
through the tissue, and parallel (nondiffused) light transmittance (%), which is thevalue of light detected without any prevention. The transmittance was deﬁned as
the diffused light transmittance divided by (100–parallel light transmittance)7.
Microscopy. Images were acquired at a Z-step of 10 μm and xy resolution of 0.794
μm using an inverted confocal microscope (×10, NA: 0.4, WD: 2.17 mm; CellVoyager™ CV1000, Yokogawa, Japan) and 488 and 561-nm lasers. Whole-brain
images were acquired using a custom-made light-sheet ﬂuorescence microscope(developed by Olympus Corporation, Japan) using a ×0.63 objective lens (NA: 0.15,
WD: 87 mm) and an sCMOS Camera (Neo 5.5, Andor Technology, Belfast, United
Kingdom) with a 1.6× digital zoom (MVX10, Olympus Corporation). Lasers withwavelengths of 488, 560, and 639 nm were used for image acquisition. Images were
captured at a Z-step of 7 μm with a pixel resolution of 6.45 μm.
Quantiﬁcation of the SN ratio and overlap ratio. Each image was semiautomatically binarized. RITC–Dex–GMA images were preprocessed using thebackground subtraction algorithm and then binarized using the triangle method.
Images of CD31 or Texas Red-conjugated lectin were preprocessed using thebackground subtraction algorithm, bandpass ﬁltered, and binarized using Yen’s
algorithm because they could not be adequately binarized with the triangle method
due to their weaker signals. Then, the binarized images were scrutinized by eye, andapparent false-positive signals were manually corrected. The overlap ratio was
calculated as the quotient of the area where the CD31-positive pixels and the
RITC–Dex–GMA or Texas Red-conjugate lectin-positive pixels were colocalized
divided by the area of CD31-positive pixels. The SN ratio of the images was deﬁned
as the Z-score of the mean intensities of the signals in the RITC–Dex–GMA or
Texas Red-conjugated lectin-positive areas calculated using the mean and thestandard deviation of the signals in the RITC–Dex–GMA or Texas Red-conjugatedlectin-negative areas in the tissue. The SN ratio of the optical sections of
CD31–EtOH–ECi-treated or SeeNet (RITC–Dex–GMA)-treated samples was calculated in the same manner. Images were preprocessed using the backgroundsubtraction algorithm and then binarized using the triangle method. Region ofinterest (150 × 150 pixels) was deﬁned in the gray matter or the white matter of the
optical slices at Z = 3150 µm.
Quantiﬁcation of PI, Iba-1, and dVenus signals. For the measurement of PI and
Iba-1 signal intensities, vascular casting of the samples was performed with
FITC–Dex–GMA, acrylamide, and VA-044. The hemispheres of the samples
were delipidated, stained, and RI adjusted. Images were taken at 200 µm deep
from the pia. The signals of the casted vessels were automatically binarizedusing the triangle algorithm, and the 2D Euclidean distances from the nearest
vessels were calculated for every pixel using the Fiji plugin. Only a few pixels
were more than 40 µm away from the casted vessels, and therefore, they were

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excluded from the quantiﬁcation. The signals of PI or Iba-1 were automaticallybinarized with the triangle algorithm, and the median intensities of the signals
in PI-positive or Iba-1-positive pixels within a deﬁned range of distances fromthe vessels were calculated. To measure dVenus signal intensities, Arc-dVenus
transgenic mice were housed under dark conditions for at least 60 h and thenexposed to an environment lined with alternating black and white stripes (2 cm
in width) on the walls at a light level of ~1500 lx. After 5 h of exposure to theenvironment, vascular casting was performed using RITC–Dex–GMA, acrylamide, and VA-044. The whole brains were delipidated, stained, and RI adjusted.
Images were taken from the deep layers of the primary visual cortex (~600 µm
deep from the pia) for quantiﬁcation.

4.

5.
6.

7.
8.

Quantiﬁcation of tissue swelling and opaque areas. Vascular casting of wildtype mice was performed using nonﬂuorescent Dex–GMA, acrylamide, and VA044. To measure the initial sizes of the brains, they were removed from the skull
before decalciﬁcation. Then, the brains were treated with EDTA, delipidated,
and RI adjusted as described above. At each time point of quantiﬁcation, thesamples were placed on a glass dish with the grid on the bottom and thenimaged using a microscope. The area of the brain was calculated in manuallysegmented images, and the linear expansion value was determined by the squareroot of the area size changes. Opaque areas were calculated in light-transmitted
images of the optically cleared samples. Speciﬁcally, the cleared samples
immersed in RI-matching solution were imaged, bandpass ﬁltered, and automatically binarized using the triangle algorithm. The original values of the
pixels that made the grids on the glass dish invisible in the binarized images
were calculated. The areas composed of pixels with values lower than theminimum values calculated above were deﬁned as opaque areas (the pixels on
the grid were not included). Then, the opaque areas were divided by the areas of
the cleared samples that were not immersed in the RI-matching solution. The
areas of the olfactory bulbs were excluded from the calculation because theolfactory bulbs were usually lost during SDS-based clearing.
Quantiﬁcation of the breakage of blood vessels. Wild-type mice underwent
vascular casting. The hemispheres of the samples were delipidated using SDS or
SDC and were immersed in ScaleCUBIC-2. Images of the neocortex were
captured using a confocal microscope. All identiﬁable penetrating vessels in adeﬁned area (~50–60 vessels per sample) were inspected by eye, and breaks inthe vessels were manually counted. Penetrating vessels were chosen for quantiﬁcation because their large size and straight morphology allowed us to estimate originally connected vessels with high conﬁdence. Pial vessels were notanalyzed because they are more vulnerable during sample handling, and a
portion of pial venules could be peeled off along with the dural venus sinuses.
Furthermore, quantitative comparisons of the breakage of pial vessels were
impractical because the majority of pial vessels were lost in SDS-treatedsamples.

9.
10.

11.

12.

13.
14.
15.
16.
17.
18.
19.
20.
21.

22.

Statistical analysis. Student’s t test was used for comparisons between two groups.
One-way or two-way analysis of variance, Kruskal–Wallis test, Tukey’s test, andSteel–Dwass test were used for comparisons among more than two independentgroups. All tests were two-sided. Statistical signiﬁcance was set at P < 0.05.

24.

Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this article.

25.

Data availability

26.

Source data underlying Figs. 1–5 is available as a Source Data ﬁle. Any additional data are
available from the corresponding author upon reasonable request.

23.

27.

Code availability
Any codes used in this study are available from the corresponding author upon
reasonable request.

Received: 21 February 2019; Accepted: 4 February 2020;

28.
29.
30.

31.

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ARTICLEand Y.I. wrote the manuscript. All authors discussed the results and commented on the
manuscript text.

Competing interests
Part of this study was performed in collaboration with the Olympus Corporation.

Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41467020-14786-z.
Correspondence and requests for materials should be addressed to T.M.

Acknowledgements
We thank Olympus Engineering for helping with the microscope design. We thank Ryuta
Koyama and Megumi Andoh for housing CX3CR1-GFP mice and Yu Sato, Kenichi
Makino and Sakura Okada for preparing materials. This work was supported by JST
ERATO (JPMJER1801 to Y.I.), JSPS Grants-in-Aid for Scientiﬁc Research (18H05525 toY.I.), the Human Frontier Science Program (RGP0019/2016 to Y.I.), AMED-CREST(AMED/MEXT to H.R.U.) and CREST (JST/MEXT to H.R.U.); Brain/MINDS (AMED/MEXT to H.R.U.); the Basic Science and Platform Technology Program for Innovative
Biological Medicine (AMED/MEXT to H.R.U.); a Grant-in-Aid for Scientiﬁc Research(S) (JSPS KAKENHI grant 18H05270 to H.R.U.); PRESTO from JST (JPMJPR15F4 to E.
A.S.); a Grant-in-Aid for Young Scientists (A) (JSPS KAKENHI grant no. 15H05650 to E.
A.S.); Grants-in-Aid for Scientiﬁc Research on Innovative Areas (JSPS KAKENHI grant
17H06328 to E.A.S.); Grants-in-Aid for Challenging Research (Exploratory) (JSPS
KAKENHI grant 18K19419 to E.A.S.); Grants-in-Aid from the Takeda Science Foundation (H.R.U. and E.A.S.); and a Grant-in-Aid from the Japan Foundation for Applied
Enzymology (E.A.S.). This work was conducted partly as a program at the International
Research Center for Neurointelligence (WPI-IRCN) of The University of Tokyo Institutes for Advanced Study at The University of Tokyo.

Author contributions
T.M. conceived and designed the study. T.M. performed all the experiments and theanalysis, except the intracranial stereotaxic injection of AAV, which was performed by
S.M. E.A.S., and H.R.U. assisted imaging by light-sheet ﬂuorescence microscope. S.Y.
provided Arc-dVenus Tg mice. A.N. and H.T. provided H-I7-iCre-imCherry mice. T.M.

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