Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

Abstract

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Mechanistic Links Between Obesity, Diabetes, and Blood Pressure: Role of Perivascular Adipose Tissue

“…In healthy endothelium, insulin has been shown to stimulate all of these microvascular properties, albeit not simultaneously. Within 10 -20 min, insulin induces vasodilation of muscle resistance arteries (456,696). Within the same time frame, insulin increases the hematocrit of the muscle microcirculation (171,716).…”

Effects of imatinib on vascular insulin sensitivity and free fatty acid transport in early weight gain

“…C1-BODIPY ® 500/510 C12(4,4-Difluoro-5-Methyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid) was from Thermo Fisher Scientific (Waltham, Massachusetts, United States) and dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 2mM [ 9 ]. Imatinib mesylate was from SelleckChem (Houston, TX) and dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 10mM [ 20 ]. Fatty acid-free bovine serum albumin (FF-BSA) was obtained from Sigma-Aldrich (St. Louis, Missouri, United States).…”

“…There is also a prominent increase in free fatty acid (FFA) levels in type 2 diabetes patients [2,18]. Elevated levels of FFAs increase their transport into organs, such as liver, pancreas, and skeletal muscles, further decreasing the perfusion of these organs and ultimately reducing insulin secretion and insulin sensitivity [18][19][20]. Increased levels of FFAs directly impair insulin mediated vasodilation [21] and trigger low-grade inflammation, which enhances endothelial permeability [22].…”

Perivascular Adipose Tissue Controls Insulin-Stimulated Perfusion, Mitochondrial Protein Expression, and Glucose Uptake in Muscle Through Adipomuscular Arterioles

“…Muscle perfusion in the thigh muscles was determined using contrast-enhanced ultrasonography (CEUS) as described (6).…”

“…After a meal, a rise in insulin induces dilation of resistance arteries within 10 min, a mechanism that regulates microvascular muscle perfusion (5,6). As a result, insulin increases microvascular blood volume (MBV) (i.e., insulin-mediated microvascular recruitment [IMVR]) in skeletal muscle (7), the primary target site for insulinstimulated postprandial glucose uptake (8).…”