Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis

With this method, one can determine what components of platelets and the hemostatic system are important for thrombosis. Additionally, this method can determine how thrombus growth and architecture are affected at the cellular level. The main advantage of this technique is that it allows a reproducible way to study many stages of thrombosis on a structural level.

A person new to this technique should avoid over-handling the tissue in the surgical area. This can lead to bleeding and can overcomplicate the identification of the carotid artery. Begin marking the injury site after exposing the carotid artery by loosely encircling the region between two surgical threads having a 0.1 millimeter size.

Place the probe under the artery distal from the lower thread. Insert the plastic piece under the artery between the two threads to mark the site for iron chloride injury. Take flow measurements before performing injury to note the basal flow.

Perform the injury by placing iron chloride-soaked filter paper on the artery for three minutes. Remove the filter paper after three minutes to prevent the variable extent of the injury and to facilitate flow counting by the probe. Add saline at the injury site to remove any excess residual iron chloride.

Monitor the flow reading. When the flow drops below 50%of the initial value, remove the probe. Quickly dry the area and add the fixative to fix the injury area externally.

Next, promptly hold the artery near the injury area with forceps. Cut downstream of the lower thread and upstream of the upper thread. Put the tissue on the plastic tissue culture dish in the same orientation as it was collected and add a few drops of the fixative.

Using a scalpel, clean the extra tissue around the artery. Ensure recording the orientation of the blood flow by cutting one end horizontally and the other tapering or oblique. Place the sample in fixative for one hour at room temperature, then store the sample in 1%paraformaldehyde at 4 degrees Celsius until sending it down wet ice for electron microscopy analysis.

The data was plotted as a Kaplan-Meier survival curve, a dot plot with bars showing the terminal blood flow at the time of either cessation of the blood flow or the termination of an experiment, or as a line graph. The blood flow increased suddenly in some cases following a gradual decrease for a few minutes indicating partial shedding of the growing thrombus and was considered an embolization event. Occlusive thrombus morphology was studied using this method as well.

The prompt collection of the sample during the thrombosis process is the most important step since delay in this process may lead to misinterpretation of our structural analysis.