FAST allowed increased sensitivity for tracking.

<p>A. FAST was able to obtain the complete track of the larva. 1. With the binary threshold method, applying a high threshold (0.09) resulted in incomplete tracks as well as noise. 2. On the other hand, applying a low threshold (0.073) resulted in noise levels too high to reliably generate complete larval tracks. 3. In contrast, using FAST with a very low threshold (0.028) produced little noise and allowed for reliable generation of complete tracks. Top row: magnified view of a single movie frame showing pixels above threshold for each method. For visualization these pixels are represented as black pixels on white background. FAST was able to isolate the larva while eliminating other noise. Arrows indicate the larva. Middle row: tracks generated using each method. Each track segment of the larva is represented by a different color. Bottom row: magnified view of middle row. B. FAST was at least 3 times faster per movie than using binary threshold method with high threshold and 20 times faster than using low threshold (n = 5).</p

Larvae in the videos were tracked using the Frame Averaging followed by Subtraction then Thresholding method (FAST).

<p>We improved video tracking by subtracting individual frames from the average of all frames. The tracking algorithm is as follows: A. For each video, calculate an average of all the frame values. B. Obtain each frame in the video. C. Calculate difference between each frame and the average frame value. D. The result was then analyzed using a binary threshold process. For better visualization the larvae are represented as dark pixels on light background for C and D.</p

Dye-fed larvae and FAST are both necessary for reliable larval tracking.

<p>A. Without dye and FAST, the larva could not be followed once it encounters the petri-dish edge. B. FAST without dye also cannot reliably follow the larva when it encounters the edge. C. Similarly, using dyed larva without FAST results in a failure to follow the larva near the edge. D. Only when both dyed larva and FAST were used in conjunction can the larvae be reliably tracked near the edge. The same movie was used between panels A., B. and C., D. Red dashed lines indicate the untracked portion of the larval track. For better illustration C and D are show as dark tracks on white background. We concluded that both the dye-fed larvae and FAST are necessary for reliable larval tracking (E).</p

Feeding larvae black dyed food enhanced contrast.

<p>A. Dyed and control larvae in 15 cm plastic petri-dish under recording camera. Top: Dyed and control larvae under low exposure. Bottom: Same larvae under high exposure, where the petri-dish edge is barely visible. Arrows indicate dyed larva. B. Dye fed larvae were visible under high exposure for up to 3 hours after extraction.</p

Complete Comparison Display (CCD) evaluation of ethanol extracts of <i>Centella asiatica</i> and <i>Withania somnifera</i> shows that they can non-synergistically ameliorate biochemical and behavioural damages in MPTP induced Parkinson's model of mice

<div><p>Parkinson’s disease remains as one of the most common debilitating neurodegenerative disorders. With the hopes of finding agents that can cure or reduce the pace of progression of the disease, we studied two traditional medicinal plants: <i>Centella asiatica</i> and <i>Withania somnifera</i> that have been explored in some recent studies. In agreement with the previous work on ethanol extracts of these two plants in mice model, we saw an improvement in oxidative stress profile as well as behavioral performance in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced Parkinson-like symptoms in Balb/c mice. Given the known potential of both the herbal extracts in improving Parkinson-like symptoms, we expected the combination of the two to show better results than either of the two but surprisingly there was no additivity in either oxidative stress or behavioural recovery. In fact, in some assays, the combination performed worse than either of the two individual constituents. This effect of mixtures highlights the need of testing mixtures in supplements market using enthomedicine. The necessity of comparing multiple groups in this study to get most information from the experiments motivated us to design a ladder-like visualization to show comparison with different groups that we call complete comparison display (CCD). In summary, we show the potential of <i>Centella asiatica</i> and <i>Withania somnifera</i> to ameliorate Parkinson’s disorder.</p></div

Microscopic confirmation of MPTP induced damage of Substantia Nigra.

<p>A. Light microscopic images show lower count of dopaminergic neurons in Substantia Nigra as shown by tyrosine hydroxylase staining. Left is brain slice from saline only treated animal and right is from MPTP treated animal. Scale is 100 μm. B. Electron microscopy shows damage induced in the nucleus of remaining SN dopaminergic neurons. Left is from saline treated animal and right is from MPTP treated animal. N label stands for nucleus. Scale is 1 μm.</p

MPTP increases lipid peroxidation (LPO) and none of the herbal extracts are able to improve lipid peroxidation levels.

<p>Left: Bar graph shows mean LPO activity of all groups. Right: CCD of different groups. Labelling scheme is similar to other bar graphs.</p

MPTP reduces both GPX and GSH levels, while all herbal extracts improve them. Herbal extracts also reduce GSH levels in saline only treated animals.

<p>Left: Bar graph shows mean GPX and GSH activity of all groups. Right: CCD of different groups. Labeling scheme is similar to other bar graphs.</p

MPTP increases catalepsy scores and decreases swimming scores. All herbal extracts are able to show improvement in MPTP treated groups.

<p>Left: Bar graph shows mean akinesia activity of all groups. Right: CCD of different groups. Labelling scheme is similar to other bar graphs.</p

MPTP reduces SOD activity, while all herbal extracts improve it.

<p>Left: Bar graph shows mean SOD activity of all groups, where error bars depict standard deviation, horizontal bars data points. Black line show only saline treated groups and blue line shows MPTP treated groups. Dotted line shows groups that are treated with herbal extracts. Right: CCD of different groups. The three values under each branch, from top to bottom are student’s T test, Tukey’s honest significant difference and Dunnett’s two tailed T test scores. Dotted right lines reflect p values similar or lower to 0.05 in Tukey’s honest significant difference test.</p