The authors extend thanks to colleagues at the University of Arkansas for Medical Sciences (Jerry Ware and Sung W. Rhee), the University of Pennsylvania (Tim Stalker and Lawrence Brass), the University of Kentucky (Sidney W. Whiteheart and Smita Joshi), and the National Institute of Bioimaging and Bioengineering of the National Institutes of Health (Richard D. Leapman and Maria A. Aronova) from whom we have learned much. The authors express appreciation to the American Heart Association and the National Heart Lung and Blood Institute of the National Institutes of Health (R01 HL119393, R56 HL119393, R01 155519 to BS and subawards from NIH grants R01 HL146373 and R35 HL150818) for financial support.

Experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Arkansas for Medical Sciences. Here, 8 - 12-week-old, wild-type male and female C57BL/6 mice were used. These mice are young adults with little accumulation of fat. The same procedures are applicable to mice mutants for various proteins important to hemostasis, such as von Willebrand factor or platelet glycoprotein VI (GPVI)5,6. All equipment, su...

Puncture Wound Hemostasis and Montaged Electron Microscopy 

<ol>
	<li>Rhee, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venous+puncture+and+wound+hemostasis+results+in+a+vaulted+thrombus+structured+by+locally+nucleated+platelet+aggregates.">Venous puncture and wound hemostasis results in a vaulted thrombus structured by locally nucleated platelet aggregates.</a> Comm Biol. 4 (1), 1090 (2021).</li><li>Pokrovskaya, I. 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We present a detailed puncture wound procedure for producing hemostatic thrombi in jugular veins and femoral arteries, their in situ perfusion fixation, and sample processing for montaged wide-area transmission electron microscopy. The overall procedures are useful for generating hemostatic thrombi for ultrastructural analysis and for comparing bleeding times in experimental mice, for example, mice treated with different types and dosages of pharmaceuticals. It is also useful for comparing bleeding times in cont...

The formation of a puncture wound thrombus that leads to bleeding cessation is one of the most essential events in life1. Yet despite that essentiality, knowledge of what occurs structurally during thrombus formation, be it in a vein, an artery, an atherosclerotic event, or an occlusive clot, has been limited by resolution and imaging depth. Conventional light microscopy is limited in depth when compared to a fully formed puncture wound thrombus, 200 to 300 &#181;m in Z1,&#160;and in resolution level when compared to the size of platelet organelles and their spacing, often less than 30 nm2. Two-photon light microscopy can yield the needed depth of imaging but does not improve resolution significantly. The most recent advances in light microscopy, for example, super-resolution techniques, are still resolution limited, in practice ~20 nm in XY and twice that in Z, and depth limited, no more than conventional light microscopy. Furthermore, super-resolution light microscopy, like much of research light microscopy, is based on fluorescence microscopy, a technique that is inherently biased to a small set of candidate proteins for which good antibodies exist or good tagged constructs3. In conclusion, conventional scanning electron microscopy can, at most, visualize the surface of the forming platelet-rich thrombus.
To overcome these technical limitations to characterizing thrombus structure, we had three goals. First, reproducibly produce a defined puncture wound in a mouse vein or artery that could then be readily stabilized in situ by chemical fixation. Second, apply a preparative procedure that emphasizes membrane preservation, a goal consistent with the aim of defining the position of individual platelets within the forming thrombus. Third, use an unbiased visualization technique that, in a single image, could be scaled between nanometer to near millimeter scale.
Montaged, wide-area electron microscopy was chosen as a major end visualization technique for a single important reason: in electron microscope imaging, one sees a vast array of features within a cell that outlines its organelles and features within the organelles. Small objects such as ribosomes can be recognized. This range of features is seen because the electron-dense heavy metal stains, uranyl, lead, and osmium, that are used for electron microscopy to yield contrast bind to a wide range of molecules. In an electron microscope image, one sees much of what is there, while with immunofluorescence and protein tagging approaches, one only sees what lights up. This means, for example, the antigen sites present on a given individual protein species. In the case of a tagged molecule, often a protein, it is the site(s) where that protein is. All other molecules are dark and not lit up. However, is this choice of electron microscopy practical? A puncture wound thrombus has a size of 300 by 500 &#181;m and, at a pixel size of 3 nm, that is an image of 100,000 by 167,000 pixels. A high-quality electron microscope camera has 4000 by 4000 pixels. That means that approximately 1000 frames must be stitched/blended to give a single image. That is a possibility that has been present in most electron microscopes manufactured in the last 15 years. The microscope stage is computerized, and the images can be stitched together with a computer. That is the rationale that led to choices underlying the formulation of the presented protocol.
Conclusively, we present below a series of steps that give a reproducible wound in the vein or artery of mice that then, following in situ fixation steps and later embedding steps, can be visualized by montaged, wide-area transmission electron microscopy at nm scale and in the stitched image visualized at near mm scale, the scale of the actual in situ fixed thrombus. Scalability of this kind is required for understanding thrombus formation as both a problem in hematology/health and as a developmental biology system in which platelets are the major cell type. These advances deliver a major virtue of electron microscopy, namely, one sees what is there, not only what lights up. For a detailed protocol on the preparation of samples for serial block face scanning electron microscopy (SBF-SEM), the reader is directed to a recent article by Joshi et al.4.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td>0.9% Normal Saline Solution</td>
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			<td>Medline</td>
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			<td>Medline</td>
			<td>NDA26393</td>
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			<td>50 mL Conical Tubes</td>
			<td>Fisher Scientific</td>
			<td>06-443-20</td>
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			<td>Alcohol Prep Pads (70% Isopropyl Alcohol)</td>
			<td>Medline</td>
			<td>MDS090670Z</td>
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			<td>HP-1M</td>
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			<td>10900</td>
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			<td>Carl Zeiss Microscopy</td>
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			<td>Medline</td>
			<td>B-D303310Z</td>
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			<td>Fisher Scientific</td>
			<td>C79-500</td>
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			<td>Corning Inc.</td>
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			<td>Medline</td>
			<td>MDS202055H</td>
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			<td>13600</td>
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			<td>Dodecenyl Succinic Anhydride/ DDSA</td>
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			<td>Dressing Forceps, 5&#34;, curved, serrated, narrow tipped</td>
			<td>Integra Miltex</td>
			<td>6-100</td>
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			<td>Dressing Forceps, 5&#34;, standard, serrated</td>
			<td>Integra Miltex</td>
			<td>6-6</td>
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			<td>EMBED 812 Resin</td>
			<td>Electron Microscopy Sciences</td>
			<td>14900</td>
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			<td>15055</td>
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			<td>Fisher Scientific</td>
			<td>03-448-17</td>
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			<td>Fisher Scientific</td>
			<td>14-649-17</td>
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			<td>Fisher Scientific</td>
			<td>7801001</td>
			<td></td>
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		<tr>
			<td>Gauze Sponges 2&#34; x 2&#34;- 4 Ply</td>
			<td>Medline</td>
			<td>NON26224H</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde (10% Solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>16120</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Liquid Inhalant Anesthesia, 100 mL</td>
			<td>Medline</td>
			<td>66794-017-10</td>
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			<td>Jeweler-Style Micro-Fine Forceps, Style 5F</td>
			<td>Integra Miltex</td>
			<td>17-305</td>
			<td>Need 2 pairs.</td>
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			<td>L/S Pump Tubing, Silicone, L/S 15; 25 Ft</td>
			<td>VWR</td>
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			<td>L-Aspartic Acid</td>
			<td>Fisher Scientific</td>
			<td>BP374-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead Nitrate</td>
			<td>Fisher Scientific</td>
			<td>L-62</td>
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			<td>Malachite Green 4</td>
			<td>Electron Microscopy Sciences</td>
			<td>18100</td>
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		</tr>
		<tr>
			<td>Masterflex L/S Easy-Load II Pump Head</td>
			<td>VWR</td>
			<td>MFLX77200-62</td>
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		<tr>
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			<td>VWR</td>
			<td>MFLX07528-10</td>
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			<td>Fisher Scientific</td>
			<td>50-191-9855</td>
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			<td>Osmium Tetroxide 4% Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>19150</td>
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			<td>Paraformaldehyde (16% Solution)</td>
			<td>Electron Microscopy Sciences</td>
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		</tr>
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			<td>Physitemp Temperature Controller</td>
			<td>Physitemp Instruments</td>
			<td>TCAT-2LV</td>
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			<td>Sigma-Aldrich</td>
			<td>P-8131</td>
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		<tr>
			<td>Propylene Oxide, ACS Reagent</td>
			<td>Electron Microscopy Sciences</td>
			<td>20401</td>
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		</tr>
		<tr>
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			<td>Fisher Scientific</td>
			<td>02-555-25B</td>
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			<td>Rectal Temperature Probe for Mice</td>
			<td>Physitemp Instruments</td>
			<td>RET-3</td>
			<td></td>
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			<td>Scotch Magic Invisible Tape, 3/4&#34; x 1000&#34;</td>
			<td>3M Company</td>
			<td>305289</td>
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		</tr>
		<tr>
			<td>Sodium Cacodylate Buffer 0.2M, pH 7.4</td>
			<td>Electron Microscopy Sciences</td>
			<td>11623</td>
			<td></td>
		</tr>
		<tr>
			<td>SomnoFlo Low Flow Electronic Vaporizer</td>
			<td>Kent Scientific</td>
			<td>SF-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SomnoFlo Starter Kit for Mice</td>
			<td>Kent Scientific</td>
			<td>SF-MSEKIT</td>
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			<td>Fine Science Tools</td>
			<td>26002-10</td>
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			<td>Carl Zeiss Microscopy</td>
			<td>495015-9880-010</td>
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		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer</td>
			<td>World Precision Instruments</td>
			<td>SYLG184</td>
			<td>silicone mat</td>
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		<tr>
			<td>Tannic Acid</td>
			<td>Electron Microscopy Sciences</td>
			<td>21700</td>
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		</tr>
		<tr>
			<td>Thiocarbohydrazide (TCH)</td>
			<td>Sigma-Aldrich</td>
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			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
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			<td>Vannas Spring Micro Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
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		</tr>
		<tr>
			<td>Von Graefe Eye Dressing Forceps, 2.75&#34;, Curved, Serrated</td>
			<td>Integra Miltex</td>
			<td>18-818</td>
			<td>Need 2 pairs.</td>
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			<td>Fine Science Tools</td>
			<td>14068-12</td>
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			<td>Braintree Scientific</td>
			<td>CLP-9868</td>
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			<td>Carl Zeiss Microscopy</td>
			<td>410135-1001-000</td>
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		</tr>
	</tbody>
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puncture wound hemostasis and preparation of samples for montaged wide-area electron microscopy analysis

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.

Author Spotlight: Revealing Platelet Dynamics Through Advances in Structural Hematology

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

We're a lab that does volume EM, structural work, to say how do platelets activate, how do they aggregate? How do they form a thrombus that causes bleeding cessation and how in the case of over stimulation, they bring about occlusive clotting. In doing this, the premise is that if we know the structure, we can then better target drugs and bring about better outcomes.We use mouse models in our research, as many researchers do, and we all have the challenge of establishing how our findings in mice and in our case in the arteries and veins in mice, carry over to the mechanisms in the much larger arteries and veins in humans. We all hope that the principles are the same in mice and humans. If you take our work and using volume EM approaches, the big things that we've contributed are one, to say that platelets are not just suicide bombers.They retain granules. They do things in response to signaling. They have stable states, and these stable states are part of a normal thrombus and they're also part of an inclusive clot.Two, we've brought the sense that there is really structure to the thrombus that forms, it's not just simply a pile of bricks, but rather it's an ordered structure in which platelets are ordered with respect to their activation, et cetera. And three, we've brought the sense that one should be able to apply and understand from structure what needs to be either stimulated to get a good outcome or what needs to be suppressed to give a outcome that will protect people from bad outcomes. We collect sequential images at near nanometer scale of a near millimeter distances.We assemble those into the single montaged image. Hence, we can observe any given spot within the image at a scale of near nanometers to near millimeter, which is sufficient to place individual features inside of the whole thrombus. Researchers now have a powerful tool, and they can ask the new question, how do coagulation cascades and cellular signals or drugs act spatially at local to global levels in thrombi?

Quantitation of drug effects on puncture wound bleeding time 
Puncture wound bleeding times provide a physiological model of a drug risk that can be readily carried out in mice. Outcomes that come from a puncture wound experiment are predictive. Here, we show a dabigatran dose-response bleeding curve. Dabigatran, a thrombin inhibitor, is used as an oral direct-acting anti-coagulant, a so-called DOAC12. The jugular vein puncture wound model was used to assess the risk inherent...

A puncture wound procedure for hemostatic thrombus formation is presented here. The formed thrombi are large and are hundreds of microns in diameter. Hence, volume imaging approaches are appropriate. We suggest montaged wide-area transmission electron microscopy as a high-resolution approach available to many and detail a preparative protocol.

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds ...

puncture-wound-hemostasis-preparation-samples-for-montaged-wide-area

Research

JoVE Journal

Medicine

Creation of a Thrombus or a Puncture Wound on a Murine Jugular Vein/Femoral Artery for Montaged Wide-Area Electron Microscopy Analysis

To begin, with a pair of scissors and blunt forceps, lift and cut the skin of an anesthetized mouse over the right jugular vein. Cut a round window in the skin large enough to expose the jugular vein and some of the surrounding tissue. Gently push aside the adipose tissue, lymph nodes, and connective tissue using the blunt dissection technique.Next, fill a 20 milliliter syringe with normal saline warmed to 37 degrees Celsius. Load the syringe into the syringe infusion pump. Then attach a 27 gauge butterfly needle and associated tubing to the syringe.Set the syringe pump to a flow rate of 0.5 milliliters per minute to wash away blood from the punctured vein or artery. Position the stream of saline a few milliliters to the side of the puncture site and not directly over it. Once the jugular vein has been cleaned, start the timer.Obtain 30 gauge and 33 gauge hypodermic needles for the jugular vein and the femoral artery respectively. Turn the bevel to face upwards. Then position the needle at a 25 degree angle over the jugular vein or femoral artery.After noting the time, quickly puncture the top of the vessel. Note the time taken for the bleeding to stop completely. Wait for the formation of a thrombus before starting perfusion fixation.Once perfusion has been completed, dissect the part of the jugular vein with the puncture site and thrombus. Use a pair of sharp scissors to make a diagonal cut across the distal end of the vessel segment and make a straight cut across the proximal end of the vessel segment. Next, place the fixed tissue at four degrees Celsius for 24 hours.The next day, pour sodium cacodylate buffer solution into a 35 millimeter culture dish containing a five millimeter thick silicone mat. Carefully transfer the tissue into the buffer, ensuring that it is submerged in the buffer. With stainless steel minutien pins and a pair of fine forceps, carefully pin each end of the vessel to the mat.Carefully clean the jugular vein. With a pair of micro scissors, cut the vessel wall opposite the puncture site lengthwise. Gently open the vessel, then pin it to the silicone mat with the intraluminal side facing up.Remove the buffer solution from the dish. Then add 1%paraformaldehyde to submerge the vessel. Immediately place the lid on the dish to prevent drying out.

159 Views.  University of Arkansas for Medical Sciences.  A puncture wound procedure for hemostatic thrombus formation is presented here. The formed thrombi are large and are hundreds of microns in diameter. Hence, volume imaging approaches are appropriate. We suggest montaged wide-area transmission electron microscopy as a high-resolution approach available to many and detail a preparative protocol. 

Puncture Wound Hemostasis and Montaged Electron Microscopy

Summary