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. 2019 Feb 4;9(1):1263.
doi: 10.1038/s41598-018-37419-4.

Astrocytic Endfeet Re-Cover Blood Vessels After Removal by Laser Ablation

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Free PMC article

Astrocytic Endfeet Re-Cover Blood Vessels After Removal by Laser Ablation

Hideaki Kubotera et al. Sci Rep. .
Free PMC article

Abstract

The astrocyte, one of the glial cells, plays many functional roles. These include provision of nutrients from blood vessels to neurons, supply of neurotransmitters and support of blood-brain barrier (BBB) integrity. Astrocytes are known to support the integrity of BBB through maintenance of the tight junction between endothelial cells of blood vessels. However, evidence of its direct contribution to BBB is lacking owing to technical limitations. In this study, astrocytic endfeet covering blood vessels were removed by the laser ablation method with two photon laser scanning microscopy in in vivo mouse brain, and the re-covering of blood vessels with the astrocytic endfeet was observed in about half of the cases. Blood vessels kept their integrity without astrocytic endfoot covers: leakage of plasma marker dyes, Evans Blue or dextran-conjugated fluorescein, was not observed from stripped blood vessels, while ablation of vascular walls induced extravasation of Evans Blue. These results suggest that the astrocytic endfeet covering blood vessels do not contribute to the immediate BBB barrier.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Astrocytes were activated by the cranial window operation on the GFAP-EGFP mice brain. Immunohistochemistry for EGFP (green) and GFAP (red) expression in cerebral cortex 10 hours (A) and two days (B) after craniotomy in one side (ipsilateral; Ipsi). The contralateral sides (Contra) are shown as a control (Ac,d,g and h). (Ae–h and Be–h) Higher magnifications of the boxes in top panels are shown. GFAP-driven EGFP was only detectable in the operated side of cortex with delay of more than one day. Filled arrowheads and open arrowheads indicate EGFP-positive/GFAP-positive astrocytes and EGFP-negative/GFAP-positive astrocytes, respectively. Scale bar, 500 µm (Ab) and 50 µm (Af).
Figure 2
Figure 2
Blood vessels were re-covered by astrocytic endfeet after ablation of endfeet. An astrocytic endfoot covering a blood vessel in the cerebral cortex of a GFAP-EGFP mouse was laser ablated. (A) Astrocytes before laser ablation. EGFP-expressing astrocytes (green) covered blood vessels labeled with EB (red). The ablated astrocytic endfoot disappeared from the blood vessel 180 min after the laser irradiation (filled arrowhead in B), and the blood vessel was re-covered by another astrocytic endfoot 360 min after (C). (Ab, Bb and Cb) Higher magnifications of the boxes in Aa, Ba and Ca, respectively. Dotted lines in Ab indicate the locations of laser ablation. Open arrowheads show the ablated astrocyte, and filled arrowhead and arrow show the stripped blood vessel and a re-covering astrocytic endfoot, respectively. Scale bar, 20 µm (Aa, Ba and Ca) and 10 µm (Ab, Bb and Cb). These images were constructed by maximum intensity z-projection of 20–50 µm depth from the brain surface.
Figure 3
Figure 3
Astrocytic endfeet re-cover blood vessels regardless of life or death of ablated astrocyte and position of the laser ablation. Laser ablation on an endfoot (A) and a stalk of process (B) of astrocytes. Astrocytic endfoot covering a blood vessel (Aa) disappeared 40 min after the laser ablation (filled arrowhead in Ab), and another endfoot of the same astrocyte re-covered the blood vessel 90 min after (arrow in Ac). (B) Laser ablation on a stalk of astrocytic process. The endfoot covering a blood vessel (Ba) disappeared 30 min after the laser ablation (filled arrowhead in Bb) and re-covered the blood vessel 22 h after (arrow in Bc). Open arrowheads point cell bodies of the ablated astrocytes. Dotted line, locations of laser ablation. Scale bar, 10 µm. These images were constructed by maximum intensity z-projections of 30–50 (A) and 20–35 µm (B) depth from the brain surface.
Figure 4
Figure 4
Schematic drawings of laser ablation of astrocytic endfeet showing patterns of astrocytic endfeet behavior after laser ablation. (A) Re-covering of stripped blood vessels irrespective of death (Aa) or life (Ab) of ablated astrocytes. (B) Stripped blood vessels were re-covered by the endfeet of ablated astrocytes (Ba) or those of other astrocytes (Bb). (C) Either endfeet (Ca) or stalks of endfeet (Cb) were laser ablated. Ablated astrocytes are colored in bright green, other astrocytes in dark green and blood vessels in red. Lightning symbols and dotted lines represent laser ablated loci, and arrowheads show removed astrocytic endfeet by laser ablation.
Figure 5
Figure 5
Extension of astrocytic endfeet during re-covering of blood vessels. An astrocytic endfoot covering a blood vessel (A) disappeared 30 min after laser ablation (arrowhead in B). (C–E) The ablated endfoot re-covered the blood vessel by extension (arrows in C–E). Dotted line, location of laser ablation. Scale bar, 10 µm. These images were constructed by maximum intensity z-projections of 10–30 µm depth from the brain surface.
Figure 6
Figure 6
Laser ablation on astrocytic endfeet did not cause BBB breakdown. (A) An astrocytic endfoot disappeared (arrowhead in Ab) one min after laser ablation (dotted line). Leakage of EB was not observed. (B) Laser ablation on vascular wall (dotted line) caused a leak of EB into the parenchyma one min after the laser ablation (Bb). Scale bar, 10 µm. These images were constructed by maximum intensity z-projections of 50–60 (A) and 35–38 µm (B) depth from the brain surface, respectively.
Figure 7
Figure 7
Astrocytes and blood vessels were labeled with SR101 and FITC-dextran. Astrocytes were labeled with SR101 (red) and blood vessels were labeled with FITC-dextran (4 kDa, green). Astrocytic endfeet covering blood vessels were laser ablated. (A) The ablated endfoot disappeared 60 min after the laser irradiation (arrowhead in Ab), and the blood vessel was re-covered by an astrocytic endfoot 360 min later (arrow in Ac). Dotted line in Aa indicates the location of laser ablation. (B) An astrocytic endfoot disappeared (arrowhead in Bb) one min after laser ablation (dotted line in Ba). Leakage of FITC-dextran was not observed. Scale bars, 10 µm. Images were constructed by maximum intensity z-projections of 60–70 (A) and 25–35 µm (B) depth from the brain surface.

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