Typical examples of neutrophil tracks and neutrophil velocity estimates.

<p>A and D: The image on the left shows a highlighted red track that is zoomed in the plot on the right, in which the centroid positions extracted from the tracking algorithm (black) and smoothed track estimate (red) are compared (the open circle indicates the track start point and the filled circle indicates the track end point). B and E: X-Y cell centroid position estimates corresponding to tracks highlighted in A and B are shown as signals with respect to time produced by the tracking algorithm (black) and estimates from the smoothing algorithm (red). C and F: X-Y velocity estimates (raw estimates in black and smoothed estimates in red), corresponding to position signals in B and E. Raw estimates of velocity were obtained by numerical differencing (central difference method) applied to the tracker position estimates.</p

Chemoattractant field inference in the zebrafish.

<p>For each zebrafish, 1–15, the estimate of the chemoattractant field (colour) is overlayed with transparency on the fish image (grayscale). Each colormap is scaled to the range −20 to 40 to provide an effective visual comparison over all fish. The chemoattractant field estimate is dimensionless hence the scale of the colormap is in arbitrary units.</p

Zebrafish experimental setup and neutrophil analysis procedure.

<p>A: Zebrafish larva from the transgenic line, Tg(mpx:GFP)i114. Neutrophils are visualised by excitation of green fluorescent protein, as previously described (Renshaw et al., 2006). The zebrafish were prepared by transection of the tailfin at the site indicated to elicit an inflammatory response, which caused recruitment of the neutrophils to the site of injury. B: The chemoattractant field inference framework. Firstly, images of neutrophil recruitment to the zebrafish wound site were acquired by video microscopy. The neutrophil centroid positions were then obtained from a segmentation and tracking algorithm. Velocities of the neutrophils were estimated from the neutrophil centroid tracks using a Kalman smoother and lastly, the velocity estimates were used in the inference of the chemoattractant field.</p

Chemoattractant field inference <i>in vitro</i>.

<p>A: Cell tracks of human neutrophils <i>in vitro</i> chemotaxing due to presence of the chemokine interleukin-8, which increases in concentration from left to right. B: Inferred chemoattractant field, normalised to the range (0,1). The chemoattractant field estimate is dimensionless hence the scale of the colormap is in arbitrary units (a.u.). C: Comparison of inferred chemoattractant field averaged over the Y-direction, to the level of chemokine interleukin-8 reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035182#pone.0035182-Tharp1" target="_blank">[30]</a>. D: Circular histogram of neutrophil angles, demonstrating a directional bias of the tracks shown in panel A towards the lower right corner.</p

Effect of maternal environment and offspring diet on the structural properties of mesenteric resistance arteries from adult (31 week-old) wild type (WT) male offspring of WT-control or Lepr^db/+ (db/+) dams.

<p>(A-D) Pressure-diameter curves of blood vessels kept under passive conditions. (E-H) Cross sectional area (CSA) of the vascular wall in mesenteric arteries kept under passive conditions at different intravascular pressures. Data are means ± SEM of n = 5–7 number of animals (vessels) per treatment group combination. *<i>P</i><0.05. SD, standard diet; HFD, high fat diet.</p

Experimental Design.

<p>(A) Animal experiments. Blood pressure (BP) and mesenteric artery structure and function were examined in male wild type (WT) offspring of WT-control and hyperleptinemic Lepr^db/+ dams. (B) Protocol to test vascular reactivity in isolated, cannulated and pressurized mesenteric resistance arteries. Two arteries were tested for each mouse. The red and green lines indicate that only one of the arteries from each mouse was exposed to either insulin or acetylcholine. At the end of each experiment all arteries were incubated in calcium-free buffer to obtain maximal passive diameters and subsequently exposed to varying levels of intraluminal pressure.</p

Effect of maternal environment and offspring diet on the morphological characteristics of mesenteric resistance arteries from adult (31 week-old) wild type (WT) male offspring of WT-control or Lepr^db/+ (db/+) dams.

<p>(A-E) Representative confocal images of mesenteric resistance arteries showing (A) nuclei; (B) F-actin; (C) elastin; (D) collagen; and (E) merged image. (F-H) Group data and representative images showing that feeding a high-fat diet was associated with a significant reduction in arterial F-actin content. (I-K) Group data and representative images showing that feeding a high-fat diet was associated with a significant reduction in arterial elastin content. (L) Vascular smooth muscle cell number, represented by nuclei contained within the medial layer of mesenteric arteries. (M) The amount of collagen contained in the wall of mesenteric arteries. Data are means ± SEM of n = 5–7 number of animals (vessels) per treatment group combination. *<i>P</i><0.05. SD, standard diet; HFD, high fat diet.</p

Effect of maternal environment and offspring diet on the mechanical properties of mesenteric resistance arteries from adult (31 week-old) wild type (WT) male offspring of WT-control or Lepr^db/+ (db/+) dams.

<p>(A-D) Strain-stress relationship curves of mesenteric arteries kept under passive conditions at different intravascular pressures. (E-H) Elastic moduli of mesenteric arteries kept under passive conditions at different intravascular pressures. (I-L) Compliance of mesenteric arteries kept under passive conditions at different intravascular pressures. The shaded areas represent the data used to calculate the elastic moduli at low pressures, which are shown in the insets. Data are means ± SEM of n = 5–7 number of animals (vessels) per treatment group combination. *<i>P</i><0.05. SD, standard diet; HFD, high fat diet.</p

Effect of maternal environment and offspring diet on mesenteric resistance artery responses to acetylcholine and insulin.

<p>All blood vessels were obtained from adult (31 week-old) wild type (WT) male offspring of WT-control or Lepr^db/+ (db/+) dams. (A-D) Acetylcholine-induced vasodilatory responses. (E-H) Insulin-induced vasodilatory responses. Data are means ± SEM of n = 5–6 number of animals (vessels) per treatment group combination. *<i>P</i><0.05. SD, standard diet; HFD, high fat diet.</p

Effect of maternal environment and offspring diet on the internal elastic lamina characteristics of mesenteric resistance arteries from adult (31 week-old) wild type (WT) male offspring of WT-control or Lepr^db/+ (db/+) dams.

<p>(A-D) Representative confocal images of the internal elastic lamina in mesenteric resistance arteries from each of the treatment group combinations. (E-G) Group data showing the number and area of fenestrae within the internal elastic lamina and the elastic modulus of elasticity normalized as a function of the percolation of the internal elastic lamina and its fenestrae. Data are means ± SEM of n = 4–5 number of animals (vessels) per treatment group combination. *<i>P</i><0.05. SD, standard diet; HFD, high fat diet.</p