LASER-ACTIVATION OF REPLACEMENT ASTROCYTES LEADS TO PRIMARY CAPILLARY CONSTRICTION

Image and Video by Tre’ Mills, PhD Candidate in the Sontheimer lab

Twitter: @astrecyte

Astrocytes extend processes called endfeet, which are thought to encapsulate more than 99% of thecerebrovascular surface(1). Due to this interaction, astrocyte endfeet are situated in a nexus that enables them to interact withendothelial cells as well as pericytes, and smooth muscle cells when present. Through the release ofangiogenic signals, astrocytes induce expression of tight junction proteins that form the blood-brain barrier(BBB), and through the release of vasoactive molecules such as Prostaglandin E2 (PGE2) can regulate localblood flow (“functional hyperemia”). Consequently, any condition that compromises the structure or function ofendfeet can cause impairments in blood flow or the BBB. In a previous study(2), we demonstrated that invadingglioma cells along the vasculature peel endfeet away, resulting in reduced expression of tight junction proteinsZonula-Occludens 1 (ZO-1) and Claudin-5 along with extravasation of various molecular weight dyes. Wefurther demonstrated(3), using the hAPPJ20 model of familial Alzheimer disease (AD), vascular amyloiddeposits aggregate between the astrocytic endfeet and the vessel wall. These amyloid-laden vessels showedan impaired ability to regulate vascular tone and blood flow upon stimulation. Given this data, we initially were interested in determining if the focal ablation of an astrocyte is sufficient to induce breaches in BBB integrity. To do so, we turned to then 2Phatal ablation method developed by Hill et al (4). Results revealed that, while focal loss of astrocyte-vascular coverage does not impair BBB integrity, it does induce a plasticity response whereby surrounding astrocytes extend processes to reinnervate vascular vacancies. In light of this exciting and novel finding, we next aimed to determine if replacement astrocytes can vasoconstrict primary capillaries. It was critical to study this particular segment of the vascular tree as astrocytes don’t regulate blood flow elsewhere (5), and this same study (5) showed that intracellular calcium rise in astrocytes through P2X1 receptors is necessary for functional hyperemia to occur. We therefore utilized Aldh1l1 cre x GCaMP5G mice to measure changes in intracellular calcium levels upon direct-laser stimulation, and intravenous injection of fluorescent dextran dyes further allowed detection of changes in vessel diameter. The new data shown here reveals that replacement astrocyte can indeed vasoconstrict primary capillaries, suggestive that they can exert physiological control over the vasculature. Future studies will aim to determine the relevance of this plasticity response in conditions with focal loss of astrocyte-vascular coverage, such as has been shown following reperfusion post-focal thrombotic stroke. 

Laser-activation of replacement astrocytes leads to primary capillary constriction 



Replacement astrocytes can vasoconstrict primary capillaries-  A far left side diagram depicts the 2Phatal sequence. An astrocyte occupying a vascular territory (top) is ablated using the 2Phatal method. Upon microglial engulfment (middle), a nearby astrocyte reinnervates that vascular vacany (bottom). To determine if this replacement astrocyte can exert physiological control over the vasculature, we followed an experimental timeline in Aldh1l1cre x GCaMP5G mice whereby a two-minute baseline was captured. We then laser activated the replacement astrocyte and observed the subsequent change in calcium response and vessel diameter. left side image is a volumetric reconstruction depicting the field at day post-ablation zero (DPA0), or baseline. The asterisks indicate ablated astrocytes and arrowhead the replacement astrocyte following ablation. The right side volumetric reconstruction depicts that exame same field 10 days later at dpa10. The arrow indicates the replacement processes from the replacement astrocyte indicated by the arrow head. these two volumetric reconstructions depict the exact same field as in b, but just from a dorsolateral view. single optical section showing the average baseline calcium level and vessel diameter during the two-minute baseline interval acquired for the experiment single optical section of the same vessel just after laser-activation of the replacement astrocyte single optical section showing that same vessel return to baseline diameter levels graph showing the change in calcium over the time-course of the experiment as quantified with the ΔF/F method graph showing the change in vessel diameter over the time-course of the experiment. The scale bar in all images is 20µm.

References

1) Watanabe K, Takeishi H, Hayakawa T, Sasaki H. Three-dimensional organization of the perivascular gliallimiting membrane and its relationship with the vasculature: a scanning electron microscope study.Okajimas folia anatomica Japonica. 2010; 87: 109-121
2) Watkins S, Robel S, Kimbrough IF, Robert SM, Ellis-Davies G, Sontheimer H. Disruption of astrocytevascularcoupling and the blood-brain barrier by invading glioma cells. Nature Communications, 5, 4196.doi:10.1038/ncomms5196
3) Kimbrough IF, Robel S, Roberson ED, Sontheimer H. Vascular amyloidosis impairs the gliovascular unitin a mouse model of Alzheimer’s disease. Brain, 138(12). doi: 10.1093/brain/awv327
4) Hill RA, Damisah EC, Chen F, Kwan AC, Grutzendler, J. Targeted two-photon chemical apoptoticablation of defined cell types in viv. Nature communications. 2017; 8, 15837. doi:10.1016/j.bbrc.2016.08.088
5) Mishra, A., Reynolds, J., Chen, Y. et al. Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles. Nat Neurosci 19, 1619–1627 (2016). https://doi.org/10.1038/nn.4428

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