Fluorescent Imaging Methodology.

<p>A grayscale white light image captured while the system’s illumination lights are on (A) while the pseudocolor image of fluorescent photon flux in the near-infrared range showing the location of TTc-related fluorescence (B). The images shown in A and B can be combined in an overlay image (C). The Region-of-Interest (ROI) measurements can be taken to quantify photon flux (D). In this example, images are from an animal 60 min after TTc-injection into the calf muscles.</p

Comparing Absolute Fluorescence values for Oxaliplatin versus Controls.

<p>Raw, uncorrected values from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045776#pone-0045776-t002" target="_blank">Table 2</a> are used in this simple T-test comparison. Notice how p-values decrease from non-significant differences to significant differences as the number of weeks increase, corresponding to the growing divergence between treated and control animals.</p

Absolute Fluorescence measurements.

<p>Observed mean fluorescence (in millions of photons per second per centimeter square per steradian (p s⁻¹ cm⁻² sr⁻¹)) measured by ROI over the spine (A). Weeks indicate the time in weeks from the start of the experiment, time in minutes indicates the fluorescence measured at that time point (post-TTc injection) on the week of the experiment. P-values are for simple T-tests comparing values at 60 minutes between week 0 and every following week. Both raw values (absolute fluorescence at 60 minutes) and corrected values (fluorescence at 60 min minus fluorescence at 0 min) are compared.</p

Relative changes in Fluorescence.

<p>Oxaliplatin dramatically decreases the amount of nerve transport that occurs to the thoracic spine. Observed mean percentage change (from t = 0) in fluorescence measured by ROI over the thoracic spine. Weeks indicate the time in weeks from the start of the experiment, time in minutes indicates the fluorescence measured at that time point (post-TTc injection) on the week of the experiment. This is the same data as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045776#pone-0045776-g003" target="_blank"><b>Figures 3A and 3B</b></a>, but in tabular form (mean +/− standard deviation).</p

Comparing Corrected Absolute Fluorescence values for Oxaliplatin vs Controls.

<p>Raw values from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045776#pone-0045776-t002" target="_blank">Table 2</a> were corrected by subtracting the baseline fluorescence at time = 0 minutes in each experiment. Simple T-tests were run and p-values are shown below. Notice that initially there is no significant differences at week 0, but that p-values become progressively smaller as week number increases, and the treated and control groups diverge.</p

Oxaliplatin dramatically decreases the amount of nerve transport that occurs to the thoracic spine.

<p>This top graph illustrates time-fluorescent uptake curves as percent change from baseline (t = 0 min) through the course of the study for oxaliplatin treated animals (n = 8/group) (<b>A</b>). The same curves for sham-treated animals in <b>B</b> where the axes are scaled identically show the decrease in TTc transport starting at 1 week, and worsening steeply from there to the end of the study. Control animals also show a decrease of uptake from about 2 weeks onward, this is likely an experimental artifact related to repeated injections to the same body part.</p

TTc transport under normal and chemotoxic conditions as demonstrated by imaging, shows impairment in Oxaliplatin-treated animals.

<p>Representative fluorescent images shows the time course of TTc uptake after left calf injection in normal (left panels) and oxaliplatin treated animals (right panels). At baseline, the uptake is identical (top panels) with migration of TTc occurring from the injection site to the spinal cord over 60 minutes. TTc fluorescent signal is always visible over the injection site in the calf, but there are significant variations in the subsequent transport of TTc between controls and treated animals. Sham treated animals maintain cord uptake similar to that seen at baseline (note the stippled ovals in the left panels at 60 minutes). During the course of oxaliplatin treatment, however, there is a progressive decrease in the amount of transport of TTc to the spinal cord (note the red stippled ovals in the right set of panels at 60 minutes). ROI measurements over the thoracic spine will yield a time-signal curve that can be analyzed for groups of animals, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045776#pone-0045776-g003" target="_blank">Figure 3</a>.</p

Oxaliplatin treatment reduces TTc transport, but not neuronal numbers.

<p>Immuno-histochemistry of representative spinal cord sections in normal (<b>A–C</b>) and oxaliplatin treated (<b>D–F</b>) animals at the S1 level. Cryosection block photographs (<b>A&D</b>), anti-NeuN immunofluorescence (B&E) and anti-TTc immunofluorescence (<b>C&F</b>) are compared and contrasted. The immuno-fluorescent anti-NeuN green fluorescence shows no difference between the control (<b>B</b>) and oxaliplatin (<b>E</b>) treated animals, indicating that oxaliplatin did not affect the number of neurons in the cord. However, the anti-TTc red fluorescence in non-treated animals (<b>C</b>) is much more prominent in comparison with the oxaliplatin treated animals (<b>F</b>), suggesting that TTc uptake and transport was affected by oxaliplatin treatment. Scale Bar = 100 µm Quantitative analysis of the histological data (<b>G</b>) (see text) shows no statistical difference for NeuN between control and oxaliplatin treated animals (p>0.56), but a significant decrease in TTc staining for oxaliplatin treated animals (p<0.0004).</p

Dynamic signal evolution across (N = 7) injections.

<p>The mean signal for lactate and pyruvate, normalized to peak carbon signal for each injection, are displayed with error bars that indicate the minimum and maximum values at each time over all injections. Total HP ¹³C was estimated by summing signal from HP ¹³C Lactate and HP ¹³C Pyruvate. The average linewidth for pyruvate and lactate peaks were 19±5 Hz and 17±5 Hz, respectively.</p

The mean, standard deviation and coefficient of variation for all repetitions (N = 7) of the dynamic phantom.

<p>Lac and Pyr refer to the total volume under the spectral and temporal curves for each tracer; <i>k_PL</i> is the forward reaction rate (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071274#pone.0071274.e006" target="_blank">Equations 6</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071274#pone.0071274.e010" target="_blank">8</a>).</p