FIGURES AND TABLES

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Protective role of reactive astrocytes in brain ischemia

Lizhen Li, Andrea Lundkvist, Daniel Andersson, Ulrika Wilhelmsson, Nobuo Nagai, Andrea C Pardo, Christina Nodin, Anders Ståhlberg, Karina Aprico, Kerstin Larsson, Takeshi Yabe, Lieve Moons, Andrew Fotheringham, Ioan Davies, Peter Carmeliet, Joan P Schwartz, Marcela Pekna, Mikael Kubista, Fredrik Blomstrand, Nicholas Maragakis, Michael Nilsson and Milos Pekny

Figure 1.

Infarct area is 2.5- to 3.5-fold larger in GFAP ^–/– Vim ^–/– (GV) than in WT mice 7 days after MCA transection. The infarct area was visualized by triphenyltetrazolium chloride and hematoxylin/erythrosin staining 7 days after proximal (A) and distal (C) transection of the MCA. (B and D) Infarct volume was more than two-fold larger in GFAP ^–/– Vim ^–/– than in WT mice (B, ^* P<0.05; D, ^*** P<0.001 and ^** P<0.01). (D) In GFAP ^–/– (G) and Vim ^–/– (V) mice, the infarct volume was not significantly different from that in WT mice. (E) Proximal MCA transection was performed at point X after bipolar coagulation at points A and B. Distal MCA transection was performed at point C (approximately 1.5 mm distal to point A). Values are means.e.m.

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Figure 2.

The territory supplied by the MCA is comparable in WT and GFAP ^–/– Vim ^–/– (GV) mice. Comparison of the line of anastomoses (A) at 2, 4, and 6 mm from the frontal pole (B). Values are means.e.m.

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Figure 3.

Astrocytes around the ischemic lesion show ET_BR immunoreactivity in WT but not GFAP ^–/– Vim ^–/– (GV) mice. (A and B) No difference was detected in the distribution of astrocytes in the penumbra in the two groups, although astrocytes of GFAP ^–/– Vim ^–/– mice showed less prominent hypertrophy of cellular processes. Green, S100; blue, cell nuclei visualized by ToPro-3. Reactive astrocytes adjacent to the ischemic lesion in WT mice (visualized by antibodies against GFAP, C and I) were ET_BR-positive (E and K) 7 days after MCA transection. (F and L) In contrast, no ET_BR immunoreactivity was detected in astrocytes in GFAP ^–/– Vim ^–/– mice. (G and M) Merged images of (C and E) and of (I and K), respectively. (C to N) Red, GFAP; green, ET_BR; blue, nuclei visualized by ToPro-3. Scale bar=50 m.

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Figure 4.

ET_BR immunoreactivity had a filamentous appearance in the cytoplasm of cultured WT astrocytes but was absent in the cytoplasm of GFAP ^–/– Vim ^–/– astrocytes, where it was often confined to the cell nuclei. Green, ET_BR. Scale bar=50 m.

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Figure 5.

ET_BR and bundles of IFs colocalize in cultured WT astrocytes. Laser-scanning confocal microscopy revealed a filamentous appearance of ET_BR immunostaining, which colocalized with GFAP-positive bundles of IFs. Red, GFAP; green, ET_BR. Scale bar=20 m.

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Figure 6.

ET_BR protein levels are comparable in WT and GFAP ^–/– Vim ^–/– (GV) cultured astrocytes. (A) Coomassie blue staining confirmed equal loading of protein. (B) Western blot analysis showed comparable levels of ET_BR protein in WT and GFAP ^–/– Vim ^–/– astrocytes. -Actin was used as a control. (C) Quantification of ET_BR protein levels from (B). (D) Quantitative real-time PCR showed higher levels of ET_BR mRNA in GFAP ^–/– Vim ^–/– than in WT astrocytes (^* P<0.05). Values are means.e.m. AU, arbitrary units.

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Figure 7.

(A) Endothelin-3 (ET3)-induced blockage of gap junction is attenuated in GFAP ^–/– Vim ^–/– (GV) astrocytes (^* P<0.05 and ^*** P<0.001). (B) Quantitative real-time PCR shows higher levels of connexin 43 (Cx43) mRNA in GFAP ^–/– Vim ^–/– than in WT astrocytes (^* P<0.05). Values are means.e.m.

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Figure 8.

Total and GLT-1-mediated glutamate uptake is decreased in the cortex of GFAP ^–/– Vim ^–/– (GV) mice. (A) Total and GLT-1-mediated glutamate uptake was lower in GFAP ^–/– Vim ^–/– than in WT mice (^* P<0.05). (B) Western blot analysis showed comparable levels of GLT-1 protein in WT and GFAP ^–/– Vim ^–/– astrocytes. Actin was used as a control. (C) Quantification of GLT-1 protein levels from (B). Values are means.e.m. (D) Glutamine synthase (GS)-positive astrocytes adjacent to the ischemic lesion in WT mice show distinct GLT-1 immunoreactivity, whereas in GV mice, GLT-1 immunoreactivity is less prominent. Arrowheads, astrocytes. Green, GLT-1; red, GS; nuclei visualized by ToPro-3. Scale bar=25 m.

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Figure 9.

Plasminogen activator inhibitor-1 mRNA levels are lower in GFAP ^–/– Vim ^–/– (GV) than in WT astrocytes cultured in the presence of 1 or 10% fetal calf serum (^** P<0.01 and ^* P<0.05). Values are means.e.m.

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