3D maximum-intensity-projection vessel rendering of a vascular cast for the same animal as Fig 4C.

<p>The box indicates tumour position whilst the arrows indicate the a. femoralis.</p

Evaluation of tumour blood flow following tumour immobilisation.

<p>DCE-MRI was performed sequentially to CT in the same mice during the same anaesthetic session and using the same cradle. Pre-contrast enhanced images were subtracted from post-contrast images at 10 minutes p.i. to reveal gadolinium-avid tumour regions. A-D: The gadolinium uptake pattern for each 8th slice of the tumour is depicted. Results for the same mouse as featured in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128537#pone.0128537.g002" target="_blank">Fig 2</a> are shown. The animal’s body is indicated by a *, its tumour by a ‡.</p

CT imaging using the proposed method.

<p>A centre slice through the tumour is shown. A: pre-contrast CT, B: post-contrast CT, C: subtraction image (post—pre). The white arrow indicates a 2-pixel discrepancy between the pre- and post-contrast image.</p

Analysis of tumour vessel diameter.

<p>The diameter of each tumour vessel was determined using a distance map approach and plotted as a histogram. A-D: Vessel diameter histograms for four representative mice, displaying a range in tumour volumes, are presented. The histograms correspond to the same tumours as portrayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128537#pone.0128537.g004" target="_blank">Fig 4</a>. The same annotations were used to identify the tumours; i.e. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128537#pone.0128537.g004" target="_blank">Fig 4</a> column A corresponds to Fig 7A.</p

Branching maps of the tumour vasculature.

<p>These are based on a skeletonization of the vesselness map segmentations and illustrate the degree of branching and the number of branches for each tumour. A-D: A showcase of branching maps for four representative mice, displaying a range in tumour volumes, is presented. The maps correspond to the same tumours as portrayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128537#pone.0128537.g004" target="_blank">Fig 4</a>. The same annotations were used to identify the tumours; i.e. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128537#pone.0128537.g004" target="_blank">Fig 4</a> column A corresponds to Fig 6A.</p

Tumour vessel image rendering.

<p>Column A-D: A showcase of four representative mice, displaying a range in tumour volumes, is presented. 3D vessel segmentations based on intensity thresholding are portrayed in the top row and are overlaid with the tumour surface. The bottom row presents a composite image of the vessel segmentations with the vesselness map segmentation. Tumour ‘C’ portrays the same animal as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128537#pone.0128537.g005" target="_blank">Fig 5</a>.</p

Inhibition of monocyte phagocytosis by anaesthetics is reversed by GABA_A antagonists.

<p><b>a</b>) Concentration dependent inhibition of phagocytosis by thiopental in primary human monocytes; <b>b</b>) In the presence of either propofol (PPF) or sodium thiopental (STP), phagocytosis was significantly restored by the addition of the GABA_A receptor antagonists picrotoxin (PTX) and bicuculline (BIC) (* sig diff. Mann-Whitney U test: p<0.05).</p

Inhibition of monocyte migration by anaesthetics is reversed by GABA_A antagonists.

<p><b>a</b>) Schematic representation of the <i>in vitro</i> transwell chemotaxis apparatus used to assay human primary monocyte migration; <b>b and c</b>) Concentration-dependent inhibition of chemotaxis was observed in the presence of propofol (PPF) and sodium thiopental (STP) (IC₅₀s  = 119 µM and 274 µM, respectively, n = 6); <b>d and e</b>) In the presence of either propofol (PPF) or sodium thiopental (STP), chemotaxis was significantly restored by the addition of the GABA_A antagonists picrotoxin (PTX) and bicuculline (BIC); (** sig diff. Mann-Whitney U test: p<0.01). Experiments were also performed with THP-1 cells and no significant differences between the behaviour of freshly-prepared human monocytes and THP-1 cells were seen (data not shown).</p

Functional responses of GABA_A receptors in THP-1 cells.

<p><b>a</b>) Typical whole-cell patch-clamp traces of THP-1 cells clamped at +60 mV in the presence of GABA and muscimol; currents were blocked by the GABA_A receptor antagonists bicuculline and picrotoxin. <b>b</b>) Application of muscimol (300 µM) to THP-1 cells revealed a reversal potential (E_rev) of 15.1 mV, and <b>c</b>) chloride-selectivity (P_Na/P_Cl) of 0.23 (n = 5). <b>d</b>) Typical Flexstation responses of THP-1 cells to different concentrations of muscimol. Buffer (continuous line) or muscimol (3, 10 and 100 µM, dashed lines) were added at 20 s. <b>e</b>) Dose curve response of THP-1 cells; EC₅₀  = 2.5 µM, n = 6.</p

mRNA and protein expression of GABA_A receptor subunits in monocytic cells.

<p><b>a</b>) Typical RT-PCR of total RNA isolated from monocyte (M) and THP-1 cell (T) lysates detecting GABA_A receptor subunits. Amplimers corresponding to the expected sizes were detected for α4, γ1 and δ subunits of the GABA_A receptor in THP-1 cells, and β2 subunits in monocytes. RNA isolated from whole human brain (B) was used as a positive control. <b>b</b>) GABA_A receptor β2 subunit expression in non–permeabilised human monocytes. Image of a human monocyte stained with Hoescht 33342 (left hand panel) to show the nucleus, and with a GABA_A receptor β2-specific polyclonal antiserum (centre) revealing cell surface β2 subunits. The right panel shows the merged image. Positive controls were human cerebral cortex and negative controls were neutrophils (data not shown). Scale bar  = 5 µm. Data are typical of at least 6 independent experiments. Inset  =  typical immunoblot of a monocyte sample (left hand side) and control (neutrophil, right hand side) probed with the β2-specific antiserum and showing expected MWt for a β2 subunit.</p