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Identification and local manipulation of bone marrow vasculature during intravital imaging.

Scientific reports (2020-04-15)
Takayuki Morikawa, Shinpei Tamaki, Shinya Fujita, Makoto Suematsu, Keiyo Takubo
ABSTRACT

Physiological regulation of blood flow in bone marrow is important to maintain oxygen and glucose supplies but also the physiological hypoxic state of the hematopoietic stem cell (HSC) niche. However, regulatory mechanisms underlying microcirculation in the bone marrow (BM) niche remain unclear. Here, we identify vessels functioning in control of blood flow in bone marrow and assess their contractility. To evaluate contractile potential of Alexa Fluor 633 (AF633; an arterial marker)-positive vessels, we performed immunohistochemistry for α-smooth muscle actin (α-SMA) and found it expressed around AF633+ vessels in the femoral and calvarial marrow. To validate AF633+ vessel contractility, we developed a simple system to locally administer vasoactive agents that penetrate BM through transcalvarial vessels. After exposure of the calvarial surface to FITC-dextran (70 kDa), FITC intensity in calvarial bone marrow gradually increased. When we evaluated the effect of transcalvarial administration (TCA) of norepinephrine (NE) on vascular tone of AF633+ arteries and behavior of transplanted blood cells, NE administration decreased artery diameter and transendothelial migration of transplanted cells, suggesting that adrenergic signaling regulates the HSC niche microcirculation and blood cell migration into the BM via effects on BMarteries. We conclude that TCA is a useful tool for bone marrow research.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Fluorescein isothiocyanate–dextran, average mol wt 70,000, (FITC:Glucose = 1:250)
Sigma-Aldrich
Fluorescein isothiocyanate–dextran, average mol wt 10,000
Sigma-Aldrich
Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody, Chemicon®, from rabbit
Sigma-Aldrich
DL-Norepinephrine hydrochloride, crystalline, ≥97% (TLC)
Sigma-Aldrich
Fluorescein isothiocyanate–dextran, average mol wt 150,000
Sigma-Aldrich
Tetramethylrhodamine isothiocyanate–Dextran, average mol wt 500,000
Sigma-Aldrich
9,11-Dideoxy-11α,9α-epoxymethanoprostaglandin F, solution, 10 mg/mL in methyl acetate
Sigma-Aldrich
FITC–DEAE–Dextran, average mol wt 70,000