Ex vivo capillary-parenchymal arteriole approach to study brain pericyte physiology

Abstract.

Significance: Vascular mural cells, defined as smooth muscle cells (SMCs) and pericytes, influence brain microcirculation, but how they contribute is not fully understood. Most approaches used to investigate pericyte and capillary interactions include ex vivo retinal/slice preparations or in vivo two-photon microscopy. However, neither method adequately captures mural cell behavior without interfering neuronal tissue. Thus, there is a need to isolate vessels with their respective mural cells to study functional and pathological changes.

Aim: The aim of our work was to implement an ex vivo method that recapitulates vessel dynamics in the brain.

Approach: Expanding upon our established ex vivo capillary-parenchymal arteriole (CaPA) preparation, we isolated and pressurized arteriole-capillary branches. Using Alexa Fluor™ 633 Hydrazide, we distinguished arterioles (containing elastin) versus capillaries (lacking elastin). In addition, our transgenic SMMHC-GCaMP6f mice allowed for us to visualize mural cell morphology and ${\mathrm{Ca}}^{2+}$ signals. Lastly, isolated microvasculature was cultured in DMEM media (up to 72 h), mounted, and pressurized using our CaPA preparation.

Results: U46619 induced a decrease in capillary lumen diameter using both a bath perfusion and local application. In addition, U46619 increased ${\mathrm{Ca}}^{2+}$ signaling both globally and locally in contractile pericytes. In our SMMHC-GCaMP6f mice, we saw that thin strand pericytes had sparse processes while contractile pericytes had long, thick processes that wrapped around the lumen of the capillary. Fresh and cultured pericytes constricted in response to U46619 to the same level, and upstream arteriolar dilation induced by capillary stimulation with 10 mM ${\mathrm{K}}^{+}$ remained unchanged by culture conditions adding another application of longer treatment to our approach.

Conclusion: Our ex vivo CaPA methodology facilitates observation of arteriolar SMC and pericyte dynamic changes in real-time without environmental factors. This method will help to better understand how mural cells differ based on microvasculature location.