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.
Highlights
Astrocytes, a type of central nervous system glial cell, play important roles for maintaining brain homeostasis, such as uptake of glutamate and GABA, provision of nutrients from blood vessels to neurons and control of extracellular pH1,2
To confirm the expression of enhanced green fluorescent protein (EGFP) in the brain of glial fibrillary acidic protein (GFAP)-EGFP mice, immunohistochemistry was performed for EGFP and GFAP on cerebral cortical sections prepared on the day and two days after the operation for cranial window
This study is the first to apply the laser ablation method to astrocytic endfeet in a mouse brain using 2PLSM. This method enabled us to reveal that astrocytes actively fill gaps in blood vessel covering created by laser ablation by using astrocytic endfeet, irrespective of whether the filling astrocyte was the target of ablation or not
Summary
Astrocytes, a type of central nervous system glial cell, play important roles for maintaining brain homeostasis, such as uptake of glutamate and GABA, provision of nutrients from blood vessels to neurons and control of extracellular pH1,2. The blood–brain barrier (BBB) is formed by tight junctions among endothelial cells, pericytes and astrocytic endfeet, and restricts entry of neurotoxins and pathogens from the bloodstream into brain parenchyma[8]. There are several tight junction proteins expressed between brain endothelial cells such as claudin-5, occludin, ZO-1 and ZO-2, which are essential to maintain BBB integrity[10]. Our previous studies suggested that activation of astrocytes is essential for recovery of BBB integrity after brain injury[16,17]. It has been suggested that the astrocytic endfoot is an integral part of BBB and regulates diffusion of solutes and water between blood vessels and brain parenchyma, direct evidence is still lacking. Mice lacking gap junction proteins connexin 43 and 30, which are enriched in the astrocytic endfoot, have dysfunction in BBB20. Aquaporin 4 (AQP4, a brain water channel) is expressed in the astrocytic endfoot facing blood vessels and that is www.nature.com/scientificreports/
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