The immediate alterations following lesions cannot be investigated by using fixed tissues. Here, we employed two-photon microscopy to study the alterations to the permeability of blood-brain barrier and to glio-vascular connections in vivo during the first minutes following cortical lesions in mice. Four models were used: (1) cryogenic lesion, (2) photodisruption using laser pulses, (3) photothrombosis, and (4) bilateral carotid ligation. Sulforhodamine101 was used for supravital labeling of astrocytes and dextran-bound fluorescein isothiocyanate for the assessment of extravasation. Transgenic mice, in which the endothelium and astrocytes expressed a yellow fluorescent protein, were also used. Astrocytic labeling in vivo was verified with postmortem immunostaining against glial fibrillary acidic protein (GFAP). Summary of results: (1) the glio-vascular connections were stable in the intact brain with no sign of spontaneous dynamic attachment/detachment of glial end-feet; (2) only direct vascular damage (photodisruption or cryogenic) resulted in prompt extravasation; (3) even direct damage failed to provoke a prompt astroglial response. In conclusion, the results indicate that a detachment of the astrocytic end-feet does not precede the breakdown of blood-brain barrier following lesions. Whereas vasogenic edema develops immediately after the lesions, this is not the case with cytotoxic edemas. Time-lapse recordings and three-dimensional reconstructions are presented as supplemental materials.
Keywords: astrocytes; in vivo imaging; photothrombosis; postlesion extravasation; supravital labeling; two-photon microscopy.