Science 13Mar2020

we acquired 2P imaging after intracisternal in-
fusion of a small CSF tracer (3-kDa dextran; Fig.
6E). During SI, tracer entered the penetrating
PVS in large quantities, reaching depths of
up to 200mm below the cortical surface and
distributing into the ISF in wild type (Fig. 6F).
Aqp4−/−mice have been shown to have abnor-
mal SD kinetics, so we first determined whether
differences in the vasodynamics of penetrating
arterioles during SI could be responsible for
the differential response in CSF influx. Knockout

animals exhibited slower arteriolar constrictions

but to a similar degree asAqp4+/+animals,

which would not explain the observed results

(Fig. 6, G to J). We then looked to see how

much traced CSF actually reached the pene-

trating PVS and how much of it exchanged

with the ISF. Less tracer entered the pene-

trating PVSs ofAqp4−/−mice compared with

their wild-type counterparts (P< 0.0001; Fig.

6K).Atthesametime,lessoftheCSFtracer

could be found in the neighboring ISF of the

penetrating PVS in the knockouts, as in previous

reports (Fig. 6L) ( 23 ). Global tracer quantifica-

tion in ex vivo brain sections confirmed a sig-

nificant reduction in fluorescence intensity in

the ipsilateral and contralateral hemisphere

ofAqp4−/−mice (P= 0.005 and 0.001, respec-

tively;Fig.6,MtoO).Inlinewiththeseresults,

AQP4 knockout animals did not develop edema

within the first 15 min after MCAO, as opposed

toAqp4+/+mice (Figs. 6P and 1H). Thus, dele-

tion of AQP4 significantly suppresses ischemia-

induced CSF tracer influx.

Discussion

Historically, brain swelling has been assumed

to develop entirely through the accumulation

of fluid from the intravascular compartment

( 15 ). The present study demonstrates that

CSF influx, initiated by ischemic SD, plays an

important role in driving acute tissue swell-

ing (Movie 10) ( 16 , 50 – 52 ). This new finding

is further supported by a detailed examination

of edema formation at CSF-ISF exchange zones,

whichweobservedincortexadjacenttolepto-

meningeal vessels as well as in periventricular

zones 30 min after MCAO in mice and during

the acute phase of human cerebral ischemic

Mestreet al.,Science 367 , eaax7171 (2020) 13 March 2020 6of15

A

SD CSF

8 8.5 9 9.5

Time after MCAO (min)

0 0.5 1

G

Time after SD (min)

J

0.0

0.2

0.4

0.6

0.8

Mean delay (min)

H

CSF

GCaMP

CSF

0.0 0.4 0.8 1.2 1.6

10

20

30

40

50

60

5

10

15

20

Time from SD onset (min)

SD speed

(μm/s)

P< 0.0001 CSF speed

(μm/s)

CSF

SD

0

20

40

60

P = 0.01

SDCSF

Max speed (μm/s)

I K

SD wave

F CSF front

Ipsilateral

369

4

6

8

10

P=0.0015

R^2 =0.94

SD onset (min)

CSF peak

(min)

0

5

10

ns

SDCSF

Time to peak (min)

D

GCaMP E

(mm

2 )

-15 -10 -5 (^051015)
0
10
20
30
0
10
20
P< 0.0001
P< 0.0001
CSF
(mm
2 )
Time after MCAO (min)
Contralateral Ipsilateral
Contra Ipsi
B C
7.76 min 8.69 min 9.61 min 15 min
MCAO
0
10
20
30
2 min
CSF
SD
Surface area
(mm
2 )
Ipsilateral
255
0
Pixel Intensit
y (AU)
Fig. 3. CSF influx is triggered by SDs after focal ischemia.(A) A fluorescent CSF tracer (BSA-594)
was delivered into the cisterna magna ofGlt1-GCaMP7 mice 15 min before MCAO and imaged using
a dual-channel macroscope for 15 min, as was done in Fig. 1D. Scale bar, 2 mm. (B) Area covered by GCaMP
and CSF tracer fluorescence. Repeated measures two-way ANOVA with Sidak’s multiple comparisons test
was performed; interactionPvalue < 0.0001;n= 6 mice. (C) CSF tracer surface area aligned to the SD
(GCaMP) peak. (D) Time to peak influx rate for the SD (GCaMP) and the CSF tracer. Paired Student’sttest
was performed. (E) Linear regression of CSF peak influx and SD onset time with 95% confidence intervals.
(F) The fronts of the ipsilateral SD wave and the CSF tracer were tracked. (G) Area covered by the SD
(left) and the CSF tracer (right) over time. (H) Front speed of the SD wave and the CSF front. Repeated
measures two-way ANOVA with Sidak’s multiple comparisons test was performed; interactionPvalue < 0.0001.
(I) Maximum speed of the SD and CSF front. Paired Student’sttest was performed. (J) Delay time
between the SD wave and the CSF tracer front. (K) Mean delay between the SD and CSF front. In (B),
(C), and (H), the shaded regions above and below the plot lines indicate SEM. In (D), (I), and (K),
error bars represent SEM.
Movie 5. Transcranial macroscopic imaging of
CSF tracer in GCaMP mice after MCAO.Dual-
channel transcranial macroscopic imaging in a
mouse that expresses GCaMP7 under theGlt1
promoter (top left) after intracisternal injection of a
fluorescent CSF tracer 15 min before MCAO (BSA-
594; bottom left). After MCAO (dashed line), there is
a rapid drop in rCBF. The surface area covered by
the fluorescent front was quantified in the ipsilateral
and contralateral hemispheres. Several minutes
after occlusion, there is a SD in the ipsilateral hemi-
sphere, triggering CSF influx. Scale bar, 2 mm.
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