The overall goal of the following experiment is to analyze endothelial calcium activity in pig coronary arteries using automated ROI analysis. This is achieved by dissecting and opening pig coronary arteries to measure endothelial calcium activity as a second step. Automated ROI analysis is performed on the acquired calcium measurements, which results in the summarized output of calcium activity.
Next software scripts are run on the output in order to statistically analyze and graphically render the results of calcium activity analysis results are obtained that show calcium signal transient parameters based on the automated ROI analysis and statistical processing. The main advantage of this technique over existing methods like manual ROI analysis is that automated analysis is quicker, more comprehensive, and less prone to user error. In this procedure, place the harvested swine right ventricles into A-P-D-M-S bottom dissection dish containing heaps buffered physiological saline solution.
Under the stereo microscope, remove a segment of the left anterior descending coronary artery by dissecting it from the surrounding cardiac tissue layers. Take care not to puncture the vessel wall. Next, place the PDMS block in the dissection dish and use a needle to pin it to the bottom.
Add buffer to the dish. Then cut an approximately 40 micron diameter tungsten wire into 12 0.3 centimeter length segments forming micro pins and place them in the dish. Use the forceps to secure one end of the vessel segment to the block with a micro pin.
Carefully insert the small spring scissors into the vessel lumen and cut longitudinally down one side of the vessel in order to open it completely. After that, orient the open vessel segment with the endothelium up. Use the remaining micro pins to secure the borders of the open vessel segment to the block such that the vessel forms a flat rectangle.
Then prepare one milliliter of flu oh 4:00 AM loading solution. Place the entire block into the loading solution for approximately 40 minutes at room temperature in the dark after loading, wash the block and heaps buffered PSS for five to 10 minutes. Subsequently, mount the block onto 50 to 100 micron thick spacers in a cover glass bottom chamber containing heaps buffered PSS.
Place the chamber on the stage of an inverted microscope equipped for confocal imaging and focus on the endothelial cell layer using confocal image sequence acquisition software capture time-lapse image sequences of basal relative fluorescence for three minutes at 20 x magnification and a frame rate of eight frames per second. After three minutes of recording basal fluorescence, replace heaps buffered PSS solution with the same volume of 100 picomolar substance P dissolved in heaps buffered PSS and record for an additional three minutes. In this procedure, render the image sequence of fluorescent calcium activity from the confocal acquisition software as an eight bit gray scale TIFF format file with no scale information.
Then open image J.Click open in the file menu and select the appropriate image sequence in the explorer window. To view the image sequence in image J, determine an appropriate ROI by using the rectangular ROI tool to estimate the upper and lower boundaries of the spatial spread of activity within the image sequence. Next, create a new folder on the computer hard drive and add the image sequence into the folder directory.
In image J, click the plugins window. Then click lc pro. To start the analysis, enter the ROI diameter value and select the filter threshold value after word.
Click the drug treatment checkbox and enter the time point values immediately before and after the drug was added. Then click okay and enter the image sequence directory into the file explorer window. In this step, download R version 3.0 0.2 and install.
Then open the 32 bit version of R.Next click file. Then open script and select the trace plot RR script for generating graphical experimental reports. Install the calibrate and gpls packages by selecting packages.
Install packages and select the appropriate packages afterward. Click file, then change directory command and use the explorer window to select the directory that corresponds to the lc pro analysis output. Then click on the script window and then the run all command to run the script here.
The time-lapse images of an input image sequence of a basal sampling interval followed by a substance P stimulated sampling interval are shown in gray scale time-lapse image sequences of best lyses for basal and stimulated intervals were rendered by lc Pro and the time dependent scaled intensity curves from each automatically positioned ROI are shown here histograms from of peak amplitude duration at half max and maximum spatial spread for both control and substance. P stimulated events from a representative experiment were rendered. Using R to graphically process, the output from lc Pro.
Notably substance P stimulation expanded the number of events and cost a significant right shift in the median amplitude and duration by a man Whitney U Test. While attempting this procedure, it's important to remember to minimize image artifact due to focal plane movement or incorrect contrast settings, as this may greatly affect analysis output.
