Paravascular microcirculation facilitates
rapid lipid transport and astrocyte
signaling in the brain
Vinita Rangroo Thrane1,2,3*, Alexander S. Thrane1,2,3*, Benjamin A. Plog1,
Meenakshisundaram Thiyagarajan1, Jeffrey J. Iliff1, Rashid Deane1, Erlend A. Nagelhus2,3
& Maiken Nedergaard1
1Division of Glia Disease and Therapeutics, Center for Translational Neuromedicine, Department of Neurosurgery, University of
Rochester Medical Center, Rochester, New York 14642,2Letten Institute of Basic Medical Sciences, University of Oslo, 0317 Oslo,
Norway,3Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, 0318 Oslo, Norway.
In the brain, a paravascular space exists between vascular cells and astroglial end-foot processes, creating a
continuous sheath surrounding blood vessels. Using in vivo two-photon imaging we demonstrate that the
paravascular circulation facilitates selective transport of small lipophilic molecules, rapid interstitial fluid
movement and widespread glial calcium signaling. Depressurizing the paravascular system leads to
unselective lipid diffusion, intracellular lipid accumulation and pathological signaling in astrocytes. As the
central nervous system is devoid of lymphatic vessels, the paravascular space may serve as a lymphatic
equivalent that represents a separate highway for the transport of lipids and signaling molecules.
with rapid fluid and solute movement1–3. Despite these impediments, the brain has a high interstitial fluid
space4,5. This anatomical space is completely ensheathed by astrocyte endfeet and is well positioned to serve as a
highway for glial-glial and glial-vascular communication1,6,7. However, the role of the paravascular space in lipid
transport and signal transduction has not been investigated in vivo. Two related questions therefore remain
unanswered: can the paravascular space facilitate rapid lipid transport and might this space act as a separate
compartment for astrocyte signaling?
facilitate diffusion of signaling molecules. In addition to the neuropil being isolated from the systemic
circulation by the blood-brain barrier, the narrow and highly tortuous extracellular space is incompatible
To outline the CSF microcirculation, tracers were infused via the cisterna magna (Fig. 1a). Both the fixable
lipophilictracerTexasredhydrazide (TXR,0.621 kDa)andthehydrophilictracertetramethylrhodamine(TMR,
3 kDa) moved rapidly through the brain along cerebral blood vessels (tracer penetration: TXR 12.57 6 4.41 and
TMR 38.71 6 7.70% brain area at 30 min). Surprisingly, lipophilic tracers of small molecular weight showed as
limited parenchymal penetration as large hydrophilic tracers (fluorescein isothiocyanate dextran, FITC,
2000 kDa) in cortical grey matter (tracer penetration 15.56 6 2.81% brain area) (Fig. 1b, c)4,5.
We used in vivo two-photon laser scanning microscopy (2PLSM) to further explore the highly selective
paravascular movement of lipophilic tracers. This restricted movement is unexpected as biologically relevant
We demonstrated that the movement of small (, 1 kDa) lipophilic tracers was highly selective to the para-
vascular space (palmitic acid, rhod-2, TXR, sulforhodamine SR101 and Oregon green BAPTA-1 OGB) (Fig. 2a,
Supplementary Fig. 1a). Intra-arterial Texas red dextran or FITC were used to morphologically distinguish
cortical surface arteries and veins as well as penetrating arterioles and venules (Supplementary Fig. 1a)4. Cross-
sectional intensity projections of penetrating arterioles confirmed paravascular tracer selectivity (Fig. 2b). By
analyzingregionsofinterestrepresenting theparavascularspaceandthesurrounding tissue(Fig.2c),weshowed
that the lipophilic tracers were rapidly cleared viathe paravascular space without gaining access to the surround-
ing tissue (normalized tracer fluorescence ratio of paravascular space to surround at 60 min: OGB 3.36 6 0.79,
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SCIENTIFIC REPORTS | 3 : 2582 | DOI: 10.1038/srep02582
SR101 3.50 6 0.88, rhod-2 4.21 6 1.35) (Fig. 2d). Deletion of the
the circulation of hydrophilic tracers in CSF, but did not affect lipo-
philic tracer movement (paravascular space to surround ratio in
Aqp42/2at 60 min: 4.83 6 1.42)4.
We next examined whether lipopohilic tracers enter and exit the
brain via similar arterio-venous paravascular routes as hydrophilic
molecules4. Using NG2-DsRed mice that have fluorescently labeled
relevant tracer palmitic acid entered via a para-arterial route
Figure 2 | Lipophilic tracers selectively enter and exit brain via paravascular space surrounding arterioles and venules. (a) Left: in vivo two-photon
image of rhod-2 circulation via the paravascular space in Glt1-eGFP mouse. White circles indicate penetrating arterioles. Surface artery (SA). Scale bar
represents 100 mm. Right: high magnification images of the paravascular space surrounding penetrating arteriole at serial depths. (b) Cross sectional
(EF). Scale bar represents 7.5 (top) and 5 (bottom) mm. (c) Region of interest (left) and analysis of tracer intensity (right) in the paravascular space and
surrounding parenchyma. Scale bar represents 10 mm. n 5 24 arterioles from 7 animals, paired t test. (d) Ratio of lipophilic tracer fluorescence in
of palmitic acid lipid along the paravascular space. Antibody against lectin outlines vascular endothelium. White arrows indicate arterioles. Scale bar
represents 100 mm. (f, g) Representative images and quantification of rhod-2 tracer in the paravascular space surrounding an arteriole, a capillary and
venule. Scale bars represent 7.5 mm. n 5 24 (arterioles), 14 (capillaries) and 12 (venules) from 7 animals, paired t test. ***P , 0.001. Data are shown as
mean 6 SEM.
Figure 1 | Rapid paravascular movement of lipophilic tracers. (a) Experimental design for studying tracer (red) movement in paravascular space via
cisterna magna. Inset: electron micrograph of penetrating arteriole (PA) with surrounding paravascular space (PVS). Scale bar represents 2.5 mm.
dextran (TMR). Top insets display auto-thresholded images. Scale bar represents 200 mm. (c) Quantification of brain parenchymal penetration.
**P , 0.01, n 5 6 animals for all groups, Mann-Whitney U. Data are shown as mean 6 SEM.
SCIENTIFIC REPORTS | 3 : 2582 | DOI: 10.1038/srep02582
tracer (rhod-2) moved sequentially in the paravascular space sur-
rounding surface arteries, penetrating arterioles, capillaries and
venules following cisterna magna infusion (normalized fluorescence
of rhod-2 to eGFP expressed under the astrocyte specific Glt1 pro-
moter: 30 min: arteriole 1.90 6 0.38, capillary 0.45 6 0.247, venule
0.23 6 0.172; 60 min: arteriole 2.34 6 0.44, capillary 0.94 6 0.21,
venule 0.44 6 0.25; 90 min: arteriole 1.74 6 0.32, capillary 0.72 6
lipophilic molecules enter the brain via para-arterial and exit via
To investigate the consequences of disrupting the paravascular
microcirculation, we temporarily depressurized the CSF compart-
ment by puncturing the cisterna magna (CMP) (Fig. 3a). Previous
studies have shown that this procedure depletes ventricular and sub-
arachnoid CSF circulation13. Using 2PLSM to image paravascular
tracer movement we demonstrate that CMP also drains all tracer
from the PVS (normalized fluorescence of rhod-2 to eGFP before
4.21 6 1.35 vs. after CMP 0.24 6 0.15) (Fig. 3b).
Since the paravascular CSF circulation appears to prevent unspe-
that CMP might accelerate lipid tracer accumulation in the par-
(such as rhod-2), which are lipophilic tracers that become concen-
trated inside cells due to their acetoxymethyl group14,15. This
that CMP accelerates intracellular accumulation of lipophilic tracer
rhod-2, when this was applied to the cortical surface or injected
intraparenchymally (eGFP normalized fluorescence of rhod-2 astro-
cyte labeling intensity at 30 min for sham control: 1.54 6 0.36 vs.
CMP: 4.01 6 0.57) (Fig. 3c, d). Conversely, Aqp4 deletion, which
slows paravascular water movement, did not enhance cellular tracer
uptake (Aqp42/2control at 30 min: 1.56 6 0.27) (Fig. 3e). Thus, an
intact paravascular space restricts lipid diffusion and cellular uptake.
Figure 3 | Depressurizing the paravascular space impairs lipid transport and astrocyte signaling. (a) Cisterna magna puncture (CMP) temporarily
depressurizes the paravascular space. Lipophilic tracer (rhod-2) was applied to cortical surface or injected into parenchyma to assess tissue influx. (b)
Cisterna magna puncture (CMP) drains nearly all paravascular tracer (rhod-2). n 5 7 arterioles from 2 animals, Wilcoxon signed ranks test. Scale bar
represents 10 mm. (c, d) Two-photon images and quantification of lipid tracer labeling in eGFP expressing cortical astrocytes (circled) following sham
control and cisterna magna puncture (CMP). Scale bars represent 75 mm. n 5 45 cells from 5 animals for both groups, unpaired t test. (e) Normalized
spontaneous calcium activity from cortical astrocytes in awake mice. Synchronized (red) and individual (green) transients. (g, h) CMP increases
frequency and reduces synchronization of astrocyte calcium signals. n 5 60 (ctrl) and 52 (CMP) cells from 10 animals (total), unpaired t test. (i–k) ATP
injection (visualized with FITC-dextran) into the paravascular space stimulates rapid and widespread astrocyte calcium wave spreading outwards from
*P , 0.05, **P , 0.01, ***P , 0.001. Data are shown as mean 6 SEM.
SCIENTIFIC REPORTS | 3 : 2582 | DOI: 10.1038/srep02582
To investigate the role of the paravascular space as a signaling
compartment, we compared spontaneous astrocyte calcium activity
in the cortex of awake mice subjected to CMP or sham surgery.
frequency and decreased synchronization of calcium signaling (ctrl
0.69 6 0.03 vs. CMP 0.60 6 0.03) (Fig. 3f–h)15. Other aspects of
astrocyte signaling were not affected (amplitude: ctrl 39.08 6 2.10%
vs. CMP 39.98 6 2.18%; duration: ctrl 21.29 6 1.29 s vs. CMP 24.74
61.62 s;P(active over15 min): ctrl75.8364.20%vs.CMP7981.05
been shown to propagate along blood vessels and the waves are
largely ATP mediated16–19. We therefore inserted a microelectrode
into the paravascular space in situ and stimulated calcium transients
space stimulated a brisk calcium wave spreading outwards from the
blood vessel, which propagated faster and over a larger area than
whenATPwasinjected intraparenchymally (wavepropagation: par-
enchyma 4.47 6 0.56 vs. paravascular space 8.89 6 1.22 mm s21;
wave diameter: parenchyma 142.86 6 12.50 vs. paravascular space
315.81 6 51.42 mm) (Fig. 3i–k).
To summarize, we show that the brain has a distinct paravascular
compartment for lipid transport and glial signaling within the nar-
row confines of the neuropil. Lipid transport follows the arterio-
venous circulation and is highly selective to the paravascular space.
Compromising paravascular transport causes increased intracellular
lipid accumulation and abnormal astrocyte calcium signaling. We
speculate that lipid transport in the brain may be spatially restricted
due to the high concentration of astrocyte-secreted lipoproteins in
CSF. Interestingly, lipoprotein mutations are the largest known risk
factor for developing Alzheimer disease20,21. Ours and previous data
therefore suggest that the paravascular compartment may represent
a lymphatic equivalent in the brain that resorbs interstitial fluid,
selectively transports small lipid molecules and can act as a signaling
highway for coordinated astrocyte communication.
Animals. Glt-1-eGFP, NG2-DsRed and Aqp42/2mice were generated as outlined
previously4,17, and mice of either sex from 6–12 weeks used in conjunction with
C57BL/6J wild-types (Jackson Laboratories) were used for experiments. All animals,
except those used for awake imaging, were anesthetized with ketamine (0.12 mg g21)
and xylazine (0.01 mg g21) intraperitoneally (i.p.). All animal experiments were
approved by the Animal Care and Use Committee of the University of Rochester.
Tracer preparation andintracisternal infusion. Thehydrophilic tracers fluorescein
isothiocyanate (FITC) dextran (0.5%, 2000 kDa) and tetramethylrhodamine (TMR)
dextran (0.5%, 3 kDa) and the lipophilic, cell-permeant tracers palmitic acid
(BODIPYHFL C161 mg ml21, 0.474 kDa, Molecular Probes), Texas red hydrazide
(0.4–2 mM, 0.621 kDa), sulforhodamine SR101 acid chloride (0.2 mM, 0.607 kDa),
rhod-2 acetoxymethyl (AM) (0.45–4.5 mM, 1.124 kDa) and Oregon-green BAPTA-
1 (OGB) AM (0.5 mM, 1.258 kDa, acquired from Invitrogen and Sigma-Aldrich)
were constituted in artificial cerebrospinal fluid (aCSF)17. These tracers were chosen
because of their small size (comparable to endogenous lipids), relevance to in vivo
imaging (e.g. as calcium indicators) and ability to cross cell membranes, such as the
endfoot membrane that encases the paravascular space15,17. The mice were secured in
a stereotaxic frame, and a 30G needle was inserted into the cisterna magna. Tracer
dissolved in aCSF was delivered at a rate of 2 mL min21over 5 minutes with a syringe
pump (Harvard Apparatus). The dyeswere used at higher concentrationsthan direct
cortical application to allow the approximate dilution factor of 155 when 10 mL was
infused into the total mouse CSF volume of 40 mL22. The cisterna magna was
punctured with a 30G needle to drain the CSF and depressurize the paravascular
space. In sham control animals the cisterna magna was exposed without puncturing
Ex vivo imaging. Mouse preparation was modified from published protocols4. The
animals were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate
buffered saline (pH 7.4) and post-fixed overnight. 100 mm vibratome brain sections
were then cut and mounted on slides using PROLONG anti-fade gold with DAPI
(Invitrogen). Epifluorescence multi-channel whole-brain montages were collected
using a virtual slice module (Microlucida Software, Microbrightfield). Exposure and
gain levels were maintained constant throughout the study. The percentage of brain
consistently thresholded images, as described previously4. For EM experiments 0.1%
glutaraldehyde was added to the perfusate/fixation solution and the ultra-thin
Lowicryl sections were prepared as outlined previously23. Images were obtained
125 mm below the surface in the barrel cortex.
a small animal ventilator (CWE), their temperature was maintained using a heating
pad, and blood gasses were collected via a femoral arterial cannula to ensure
physiological hemodynamic parameters17.Tovisualize the cerebral vasculature FITC
or Texas red dextran (Invitrogen) were administered intra-arterially. A steel frame
was secured to the skull using dental cement, and a 2 mm craniotomy was opened
over the somatosensory cortex with particular care being taken not to puncture the
duramater. Tostabilize imaging, the craniotomywasthensealed withagarose(1.5%,
type III-A, Sigma) and a coverslip. A Mai Tai laser (SpectraPhysics) attached to a
confocal scanning system (Fluoview 300, Olympus) and an upright microscope
(IX51 W) were used. Tracers and eGFP were excited at 850–890 nm and emission
wascollected at 575–645 nmusing a20x (0.95NA) lens. 5123512 pixel frames were
collected from the pial surface to 200 mm depth at 20 mm z-steps. Superficial arteries
and veins were distinguished based on morphology (e.g. arteries pass more
superficially, and have fewer branches near the surface)4. Tracer movement was
analyzed as outlined previously by defining doughnut shaped ROIs around cerebral
Awake calcium imaging. Animal preparation was performed as described by the
authors previously15. Briefly, mice were anesthetized with isoflurane (1.0–1.5%),
head-restrained with a steel mini-frame, and habituated to imaging through training
was loaded onto exposed cortex before applying the coverslip. Calcium signaling was
at 0.2 or 1 Hz. Calcium transients were analysed using previously described custom-
made software (MatLab Inc.) and Image J (NIH)15,17.
In situ calcium imaging. Acute cortical slices were prepared from P10-20 mice as
described previously16,17. Briefly, 400 mm acute cortical slices were incubated with
rhod-2 (2 mM) for 20 min, before being transferred to a recording chamber where
they were imaged and analyzed as outlined before17. ATP (500 mM in aCSF) was
injected via a glass microelectrode 40–80 mm into the paravascular space or
parenchyma of the slice using a picospritzer (10 psi, 100 ms, Parker
Instrumentation). We used FITC dextran (1%, 2000 kDa) to visualize the injection.
Statistical analyses. All analysis was performed using IBM SPSS Statistics 19 and all
tests were two-tailed where significance was achieved at a 5 0.05 level. Wherever
necessary a Bonferroni correction for multiple testing was done.
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We thank J. Rothstein and O.P. Ottersen for providing the transgenic animals, and G.A.
Gundersen for the electron micrograph. This work was supported by the US National
Institutes of Health (NS075177 and NS078304 to M.N. and F31NS073390 to N.A.S.),
Research Council of Norway (NevroNor, and FUGE grants), Letten Foundation, and
V.R.T., A.S.T., E.A.N. and M.N. planned the project, prepared figures 1–3 and wrote the
main manuscript text. V.R.T. and A.S.T. performed in vivo and in situ experiments. B.P.,
M.T., J.J.I. and R.D. performed immunohistochemistry and contributed to the manuscript
text and figures. All authors reviewed the manuscript.
Supplementary information accompanies this paper at http://www.nature.com/
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Rangroo Thrane, V. et al. Paravascular microcirculation facilitates
rapid lipid transport and astrocyte signaling in the brain. Sci. Rep. 3, 2582; DOI:10.1038/
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