Science - USA (2020-03-13)

Mestreet al.,Science 367 , eaax7171 (2020) 13 March 2020 10 of 15

N

CSF tracer

AQP4

-15 -10 -5 0 5 10 15

0

5

10

Time after MCAO (min)

Aqp4+/+

Aqp4-/-

P<0.0001

Ipsilateral CSF

(

F/F

) 0

MCAO

0 min

CSF tracer

Contra Ipsi

A

4 min

255

0

Pixel Intensity (AU)

6 min 8 min

Aqp4

+/+

Aqp4

-/-

B

0 min 6 min 8 min 10 min

Aqp4+/+ Aqp4-/-

0

2

4

6

8

Time to peak (min)

ns

500

600

700

800

900

1000

Mean pixel

intensity (AU)

P=0.0048

P=0.0011

Aqp4+/+ Aqp4-/-

Contralateral Ipsilateral

Aqp4+/+ Aqp4-/-

Contralateral Ipsilateral

M

O P

Cortical surface

(^0) Fmax F15 min
5
10
F/F
0
P=0.0180
ns
Ipsilateral -100
-50
0
50
P<0.0001
Aqp4+/+ Aqp4-/-
0.5 min
d/d
SI
I
MCAO
2P
CSF tracer
i.v. dextran
CSF tracer
G
-0.9 min 2.7 min 4.4 min
SI ytis
netnilexi
)
P
UA(
0
255
CSF tracer
-100 μm
Penetrating
PVS
C D
E H
1
2
3
4
PVS
0.5 min
P<0.0001
Aqp4+/+ Aqp4-/-
Interstitial
fluid (ISF)
(
F/F
) 0
Aqp4+/+
J
K
3.7
3.8
3.9
4.0
Cortical water content (ml g dry weight
-1
) ns
MCAO Sham
Aqp4
+/+
Aqp4
-/-
0
500
i.v. dextran CSF tracer
re
car
tFSC
)
UA
(
SI
PVS
SI
ISF
-100 μm
0.5 min
ISF
(
F/F
SI
)
P<0.0001
0.5
1.0
1.5
2.0
i.v. dextran
Art. diameter
(
)
2P
CSF tracer
L
MCAO Spreading ischemia (2.7 min)
1.6 min
F
5.1 min
Pial PVS
Capillary influx
0
-202 μm
Penetrating PVS
1.5 min 5.0 min
0
-202 μm
Spreading ischemia (3 min)
Aqp4
+/+
Aqp4
-/-
Fig. 6. CSF-mediated edema after MCAO is dependent on AQP4 expression.
(A) Transcranial imaging after intracisternal tracer injection (BSA-647) inAqp4+/+
andAqp4−/−mice. (B) Quantification of ipsilateral CSF tracer influx (DF/F 0 ).
Two-way repeated measures ANOVA was performed;Pvalue from interaction term
<0.0001;n= 4 or 5 mice per genotype. (C) Time of CSF influx peak. Unpaired
Student’sttest was performed. (D) Change in maximumDF/F 0 (Fmax)to15min
after MCAO (F15 min). Paired Student’sttest was performed. (EandF)Volumetric
2P imaging shows CSF tracer (3-kDa dextran) entering the brain via the surface
and penetrating PVSs inAqp4+/+andAqp4−/−mice. After SI, the tracers were
also found surrounding capillaries only inAqp4+/+.(G) Time-lapse 2P imaging of a
penetrating arteriole 100mm below the cortical surface. (H) The tracer enters
the ISF after SI. (I) CSF tracer entered the penetrating PVS of bothAqp4+/+and
Aqp4−/−mice. The dashed circles indicate the regions of interest used for the
quantificationsin(K)and(L).(JtoL) Penetrating arteriolar (art.) diameter changes
after MCAO (J). Tracer influx was quantified in the penetrating PVS (K) and
the ISF neighboring the same PVS (L). One PVS per mouse was analyzed;n=4or
5 mice per genotype. (MandN) Immunohistochemical labeling for AQP4 from
ipsilateral dorsal cortex ofAqp4+/+(M) andAqp4−/−(N) after CSF tracer
injection. (O) Mean pixel intensity from six coronal sections from 4 or 5 mice per
genotype. (P) Cortical water content 15 min after MCAO or bilateral cortices
from sham-treatedAqp4−/−mice. Paired Student’sttest was performed;n=5
or 6 mice per group. Scale bars, 2 mm (A), 50mm (F) to (I), and 500mm
(M) and (N). In (B), (J), (K), and (L), the shaded regions above and below the
plot lines indicate SEM. In (C), (O), and (P), error bars represent SEM.
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