Measurement of urinary catecholamines in small samples for mice.

INTRODUCTION: Analysis of catecholamines in small samples of urine is difficult and sensitive to stress. Current techniques require pooling of samples or expensive separation by double mass spectrometry. A method for extraction of unconjugated catecholamines in 20µL urine samples has been developed using alumina extraction prior to separation by high performance liquid chromatography (HPLC) and electrochemical detection (ECD). METHODS: Three murine experiments tested the application of the procedure. In the first, collection occurred in the morning and evening prior to handling, and in the morning after three days of handling. In the second, passively obtained urine was compared to stressfully obtained urine in the same mice. Finally, basal collections were compared to urinary catecholamine levels 15 and 30min into novel cage stress. Urine was extracted alongside 2,3-dihydroxybenzoic acid (DHBA) internal standard via alumina and brought to pH 8.5 with tris buffer. The mixture underwent two wash steps for depuration and eluted with perchloric acid for analysis on HPLC with ECD. RESULTS: This novel extraction method using low amounts of urine yielded 48% recovery in the samples and 60% recovery in the standard extraction on average. With a signal to noise ratio of 3:1, the limit of detection (LOD) of a standard is 1.2pg/mL, which allows for the detection of 3.6pg/mL in urine or 72fg in a 20µL sample. Thus resting catecholamine levels are 216 times higher than the LOD. Unconjugated norepinephrine and epinephrine levels were significantly increased 15min after novel cage stress and epinephrine remained elevated after 30min, but did not show significant differences when comparing collection time, handling exposure, or specific collection technique. DISCUSSION: The technique is an effective measure for sympathetic activity in micro samples, with a limit of detection in the attomole range for 20µL samples.

Quantification of plaque neovascularization using contrast ultrasound: a histologic validation.

AIMS: The density of vasa vasorum within atherosclerotic plaque correlates with histologic features of plaque vulnerability in post-mortem studies. Imaging methods to non-invasively detect vasa vasorum are limited. We hypothesized that contrast ultrasound (CUS) can quantify vasa vasorum during atherosclerosis progression. METHODS AND RESULTS: New Zealand white rabbits received a high-fat diet for 3 weeks, and bilateral femoral artery stenosis was induced by balloon injury. Contrast ultrasound femoral imaging was performed at baseline and 2, 4, and 6 weeks post injury to quantify adventitial videointensity. At each imaging time point 10 vessels were sectioned and stained with haematoxylin and eosin and von-Willebrand factor. Adventitial vasa vasorum density was quantified by counting the number of stained microvessels and their total cross-sectional area. Plaque size (per cent lumen area) progressed over time (P < 0.001), as did adventitial vasa vasorum density (P < 0.001). Plateau peak videointensity also progressed, demonstrating a strong linear correlation with histologic vasa vasorum density (P < 0.001). Receiver operating characteristic analysis indicated that a three-fold increase in median adventitial videointensity had a sensitivity of 100% and specificity of 88% for predicting abnormal neovascularization. CONCLUSION: We have histologically validated that CUS quantifies the development of adventitial vasa vasorum associated with atherosclerosis progression. This imaging technique has the potential for characterizing prognostically significant plaque features.

A novel hydrodynamic method for microvascular flow enhancement.

We have shown that drag-reducing polymers (DRP) restore perfusion to a stenotic bed by lowering microvascular resistance. We studied whether resistance-lowering by DRP are due to changes in hydrodynamics or vasodilation. During intravital microscopy of rat cremaster muscle (n=18), DRP infusion increased aortic flow (p<0.002), decreased vascular resistance (p<0.01), increased arteriolar diameter (p=0.023), and increased RBC velocity in the arterioles (p<0.04), venules (p<0.003) and capillaries (p<0.02). To investigate whether DRP lowers resistance without involvement of shear (nitric oxide [NO])-mediated vasodilation, L-NAME was infused in 19 rats, but failed to abolish DRP resistance-lowering. To further investigate whether DRP resistance-lowering depends on vasodilation, adenosine was infused into rabbit femoral arteries (n=19) prior to DRP to achieve marked vasodilation. DRP caused an additional 14% decrease in femoral vascular resistance (p=0.022). DRP enhance microcirculatory perfusion by lowering vascular resistance. This involves not only some degree of shear-induced vasodilation, but also tone-independent resistance lowering mechanisms, suggesting that DRP favorably alter blood flow hydrodynamics. Modulation of blood flow hydrodynamics to enhance perfusion is unique, and may be of therapeutic value for any condition of compromised blood flow.

Fate of culture-expanded mesenchymal stem cells in the microvasculature: in vivo observations of cell kinetics.

Vascular delivery of mesenchymal stem cells (MSCs) following myocardial infarction is under clinical investigation. Little is known about the microvascular fate of MSCs. We used intravital microscopy of rat cremaster muscle microcirculation to track intraarterially delivered MSCs. Rat MSCs (average diameter, 23 microm) were bolused into the ipsilateral common iliac artery. Interrogation of an arteriole-venule pair revealed that 92+/-7% (n=6) of MSCs arrest and interrupt flow during first pass at the precapillary level, resulting in decreased flow in the feeding arteriole (velocity decreased from 6.3+/-1.0 to 4.6+/-1.3 mm/sec; P<0.001). MSC deformability evaluated using filtration through polycarbonate membranes revealed that the cortical tension of MSCs (0.49+/-0.07 dyne/cm, n=9) was not different from that of circulating mononuclear cells (0.50+/-0.05 dyne/cm, n=7). When intravital microscopy was performed 3 days following injection, the number of MSCs in the cremaster further decreased to 14% of the initial number, because of cell death in situ. In vivo labeling of the basement membrane revealed that at 1 day, the surviving cells were spread out on the luminal side of the microvessel, whereas at 3 days, they integrated in the microvascular wall. Despite their deformability, intraarterially delivered MSCs entrap at the precapillary level because of their large size, with a small proportion of surviving MSCs integrating in a perivascular niche.