Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis

Abstract

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.

Figure 1:. Surgical set-up to perform a jugular vein/femoral artery puncture wound thrombus experiment.

Surgical equipment (A) and instruments (B) are carefully arranged for efficiency of movement since timing is critical during the experiment. (C) After perfusion fixation, the portion of the vessel containing the puncture wound thrombus is pinned to a silicone mat in a 35 mm culture dish while viewing under the light microscope.

Figure 2:. Going from a blood vessel to plastic embedded samples that will be sectioned for either WA-TEM or SBF-SEM.

(A) A femoral artery with a puncture wound and accumulating extra-vascular thrombus formation (circle and arrow) is shown. (B) A thrombus (circle and arrow) is embedded in plastic for sectioning for WA-TEM with a microtome for subsequent 2D imaging. (C) A thrombus (circle and arrow) is embedded in plastic, trimmed to give a short stick, less than 1 mm, for attachment to a pin within an SBF-SEM imaging chamber.

Figure 3:. Puncture wound to thrombus formation:

A schematic of preparative flow from a puncture wound (A) to an image slice of a 1 min post-puncture jugular vein thrombus (B). The vessel wall is labeled in blue on the image slice, the small black dots near the vessel wall are red blood cells, and the thrombus platelet aggregates appear as dark gray areas. This figure has been modified from.

Figure 4:. An example of data, in graphic form, generated in a jugular vein puncture wound experiment.

A dabigatran dose-response bleeding curve is shown. This figure has been modified from. The bleeding time means at various concentrations of dabigatran are shown along with error bars indicating plus or minus standard deviation. Statistical analysis using the student’s t-test indicated a p-value of < 0.05.

Figure 5:. WA-TEM images of a slice stitched together to form a montage.

(A) Individual high-resolution frames are collected and then stitched together for the purpose of placing features in a larger context. (B) By zooming up, important details can be brought out in context (poly: polymorphonuclear leukocyte, endo: endothelium, and col: collagen fibers).