Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

The overall goal of the following experiment is to visualize the typic interaction between platelets and neutrophils on the activated endothelium during vascular disease. This is achieved by real-time fluorescence intra vital microscopy in live mice to visualize the microvasculature within the cremaster muscle. As a second step fluorescently labeled antibodies are infused and ular inflammation or injury is initiated to facilitate direct visualization of the resulting typic platelet neutrophil interactions.

Next, the saved data are analyzed in order to quantify the number of cells and their dynamics. Ultimately, direct typic interactions between platelets and neutrophils on the activated endothelium can be assessed based on the fluorescent signal of the infused antibodies in real-time intravital microscopy. Our intravital microscopic technique can provide insight into the real-time interactions between platelets and neutrophils on activated endothelium during vascular inflammation and thrombus formation.

This technology can also be applied to other systems such as evaluating the interactions of cancer cells and blood cells. Before starting the experiment, turn on the microscope system for the vascular inflammation model. Intra growly inject mirroring TNF alpha three hours prior to IP injection of anesthetics.

Once sedation has been confirmed by toe pinch cannulate a PE 90 tube into the mouse's trachea to eliminate any breathing difficulties. Next, cannulate a PE 10 tube into the left jugular vein for the infusion of fluorescently labeled antibodies and additional anesthetics. Then gently pull out and horizontally inci the scrotal skin and suse prewarm buffer over the surgical field.

Now pressing on the lower abdomen with one forcep, carefully pull out a testicle with the other forcep in size, the car master muscle vertically and flatten the muscle over a glass cover slip on an intra vial microscope tray by pinning the peripheral tissue to the tray. Begin this step by first infusing DITE 4 88 conjugated rat, anti mouse CD 42 C and Alexa floor 6 47 conjugated anti mouse GR one antibodies through the previously set jugular cannula. Then in the filter set of the microscope, mark the checkbox for the channels that will be exposed and set the exposure time for each select time-lapse capture.

To set the number of time points to 1000 to 1500 with an interval of 200 milliseconds for a total elapsed time of five minutes. Once all of the parameters have been set in the image capture, select start to begin the capture capture at a 60 x magnification with a 157 micrometer by 118 micrometer window. Monitor the ruling and adherent platelets into neutrophils for five minutes in the top half of an inflamed cremaster venue.

To analyze the platelet thrombus formation, go to mask and select create. Then under the marquee tool in the main view, click the large pencil icon. Use the pencil to color a region outside the vessel in the main view.

To set a background mask, go to mask, click copy this plane, click copy mask. In current time point, select all time points and click okay. To calculate the background signal, go to statistics and click mask statistics.

Then in image scope, click current 2D time-lapse, or four D images in mask scope, select entire mask in features. Under date of capture select elapse time. Under morph geometry, select area in pixels and under intensity select maximum intensity.

Now click export. Open the text file in a spreadsheet program and calculate the average value of the background signal throughout the recording period. To determine the background fluorescence intensity.

After calculating the background fluorescence intensity during the platelet thrombus as just demonstrated in the ular inflammation model, go to mask, click segment, select fit e, and channel, and insert the average value of the background signal on low click apply and okay. Then go to statistics and click mask statistics. Now in image scope, click current 2D time-lapse or four D images in mask scope, select entire mask in features under date of capture select elapsed time under morph geometry, select area in pixels and under intensity, select some intensity.

Then click export. Open the text file in the spreadsheet program and calculate the fluorescence intensity of the platelet thrombus. For analyzing arteriolar thrombosis begin by infusing DITE 4 88 conjugated anti mouse CD 42 C ANDOR 6 47 conjugated anti mouse GR one antibodies as just demonstrated.

After clicking start to initiate the image capture process, double click on a spot two to three micrometers internally from the vessel wall to fire the laser. The injury of the arteriolar endothelial cells causes a visual shape change that should be apparent in the bright field image followed by platelet accumulation. Five minutes after the laser injury, pause and cancel the capture.

Subsequently start a capture with 2, 400 time points with a 500 milliseconds interval to record the adherent platelets and rolling and adherent neutrophils for 20 minutes. After calculating the background fluorescence intensity during the platelet thrombus in a similar manner as in the ular inflammation model, go to mask, click segment, select fite and channel and insert the average value of the background signal on low click apply and okay. Now in image scope, click current 2D time-lapse or 40 D images in mask scope, select entire mask in features under date of capture select elapsed time under Morpho select area in pixels and under intensity, select some intensity.

Then click export. Open the text file in the spreadsheet program and calculate the fluorescence intensity of the platelet thrombus. To quantify the rolling and adherent neutrophils, play the time lapse and count the number of cells that visibly roll over.

The platelet thrombus over 20 minutes rolling is defined as a decrease in neutrophil speed while interacting with the platelet thrombus for at least two seconds. Adherent neutrophils are defined as any neutrophils that remain attached to the platelet thrombus for at least two minutes. Here, representative ized images of the appearance of fluorescent signals associated with neutrophils in red and platelets in green.

During ular inflammation is shown, the arrow shows the direction of the blood flow in the vessel. The number of rolling and adherent neutrophils on inflamed endothelial cells was determined to be 0.25 cells per minute and 18.5 cells per five minutes respectively. The data are representative of the mean plus or minus the standard error of the mean of 30 different Es in four wild type mice.

Here the median integrated fluorescent signal of the platelets is plotted as a function of time. It was found that most platelets in red adhere to the adherent and crawling neutrophils in green rather than to the inflamed vessel wall. Now moving to neutrophil platelet interactions following laser induced arteriolar injury.

These images illustrate a single neutrophil in red and indicated with the yellow arrow rolling over the platelet thrombus in green with a second neutrophil in red and indicated by the white arrow rapidly rolling over the arteriolar endothelial cells, both over a five second capture interval here, a single neutrophil again in red is shown rolling over and adhering to a platelet thrombus in green over a 15 second interval. The arrowhead identifies the rolling and a adherent neutrophils and the thick gray arrow indicates the direction of the blood flow. Here, the median integrated fluorescent signal of the platelets is plotted as a function of time.

The number of rolling and adherent neutrophils was determined to be 21.5 and 1.6 cells over 20 minutes respectively. Initial rapid rolling of the neutrophils occurred on the endothelial cells. Once the neutrophils contacted the platelet thrombus, the rolling velocity of neutrophils on the platelet thrombus was changed with a range of 8.2 micrometers per second and is mediated by the interaction of pectin and psgl L one.

The data are representative of the mean plus or minus, the standard error of the mean of 14 different thrombi in seven wild type mice five minutes after laser injury. The size of the platelet thrombus remains relatively constant during imaging, and the neutrophils roll onto and adhere to the thrombus. The fluorescent signal from the circulating platelets is negligible.

In comparison with that from the platelet thrombus, After watching this video, you should have a good understanding of how to set up the intravital microscope system with live mice, allowing you to visualize in real time the hetero typic interactions between platelets and neutrophils on activated endothelium within the microvasculature.