This work was funded by the National Institutes of Health, Grant R01HL13334. The authors would like to thank Dr. Kevin Haworth for assisting with Drabkin&#39;s assay and Dr. Viktor Bollen for his support in designing the protocol. The authors are also thankful to Dr. Adam Maxwell for his guidance on designing the histotripsy source.

For the results presented here, venous human blood was drawn to form clots after approval from the local internal review board (IRB #19-1300) and written informed consent provided by volunteer donors24. This section outlines&#160;a design protocol to assess lysotripsy efficacy. The protocol is based on a previous work by Bollen et al.24.
1. Clot modeling
NOTE: Prepare the clots within 2 weeks but more than 3 days prior to the day of the experiment to ensure clot stability and maximize retraction28. Prepare the clot following the approval from local institutional review board.
<ol>
	<li>Prepare borosilicate Pasteur pipette for storing the blood (see Table of Materials for specifications of the pipette). Borosilicate tubes are used because of the hydrophilic nature of the material which promotes platelet activation and clot retraction29. Seal the tip of the pipette via heating over a Bunsen burner.</li>
	<li>Draw fresh human venous blood. Aliquot the total blood drawn in 2 mL increments per desired clot. Transfer each 2 mL aliquot to one Pasteur pipette. 
	NOTE: Execute step 1.2 within approximately 3 min of blood draw so that blood does not clot before transferring to pipettes.&#160;Also, ensure the volunteer donor is not on any medications that may alter the clotting cascade (e.g. blood thinners or platelet inhibitors).</li>
	<li>Incubate the blood aliquots within the pipettes (equal to the number of clots required) in a water bath for 3 h at 37 &#176;C.</li>
	<li>Store the pipettes for a minimum of 3 days at 4 &#176;C to allow for retraction of clots28. As the clots retract, serum will be observed to accumulate at the top of the clot within the pipette. The rt-PA response of clots remains stable for 2 weeks following retraction28.</li>
</ol>
2. Water tank preparation
<ol>
	<li>Fill the water tank with reverse osmosis water. Line the bottom surface of the tank with an acoustic absorbing material to reduce reflection of the therapy or imaging ultrasound pulses. Use a water handling system to degas and filter the water to minimize bubble nuclei. 
	NOTE: One way to filter the water is using an inline filter. The specifications of the bag used for generating the representative results is given in the Table of Materials.</li>
	<li>Place two heating elements at the bottom surface of the tank. Heat the water to 37.3 &#730;C to achieve the maximum lytic enzymatic activity30.</li>
	<li>Setup a flow channel as shown in Figure 1A. The flow channel consists of tubing, a model vessel with material and geometric properties representative of an iliofemoral vein, a reservoir for plasma, and a syringe on the distal most end of the reservoir (Table of Materials). The syringe is connected to a pump to regulate a flow through the channel during the experiment.</li>
	<li>Manually submerge the flow channel in the water tank to bring the channel to physiologic temperature during the degassing/filtering/heating stage (steps 2.1 and 2.2).</li>
</ol>
3. Preparation of plasma and rt-PA mixture
<ol>
	<li>Prior to experiment day 
	&#8203;NOTE: When stored at -80 &#176;C, plasma is stable for at least 2.5 years31 and rt-PA is stable for at least 7 years32. Therefore, execute step 3.1 anytime within these periods to ensure stability of the two components.
	<ol>
		<li>Dilute the rt-PA obtained from a manufacturer in powdered form to 1 mg/mL in sterile water.</li>
		<li>Aliquot 100 &#181;L of diluted rt-PA into 0.5 mL centrifuge tubes and store them at -80 &#176;C.</li>
		<li>Aliquot 35 mL of human fresh-frozen type O plasma in 50 mL centrifuge tubes. Store the tubes at -80 &#176;C.</li>
	</ol>
	</li>
	<li>On the experiment day
	<ol>
		<li>Retrieve plasma aliquots from the freezer. Retrieve as many aliquots as the number of clots to be tested that day. Immerse the frozen aliquots in a water bath at 37 &#176;C to thaw (~10 min).</li>
		<li>Once plasma has thawed, pour it into a beaker that is triply rinsed with ultrapure water. Lightly cover the mouth of the beaker with aluminum foil to prevent contamination and place the beaker in the water bath. Allow the foil to be loose enough to allow air to contact the plasma.</li>
		<li>Let plasma equilibrate at 37 &#176;C to atmospheric pressure for at least 2 h.</li>
		<li>Take out the frozen rt-PA vials and place on ice until needed, with one vial for each experiment run.</li>
		<li>Make low gelling agarose (2%) in a 50 mL flask, by dissolving agarose in ultrapure water. Choose the total amount of agarose solution such that approximately 2 mL is available for each specimen to be analyzed. Heat the solution in flask in a microwave until bubbly. Secure the flask with a waterproof screw lid on it. Submerge the flask in the water bath alongside the plasma. 
		&#8203;NOTE: This step ensures agarose is available to secure exposed clot segments for histology analysis following histotripsy insonation.</li>
	</ol>
	</li>
</ol>
4. Setting up histotripsy source and imaging array
<ol>
	<li>Ensure the motorized positioners can be controlled from the runtime environment of a programming platform, using directions and commands provided by the manufacturer. Check that the motors of the system are connected to the appropriate port of the computer with the runtime environment.</li>
	<li>Mount the histotripsy source on the motorized positioning system as shown in Figure 1B.</li>
	<li>Connect the histotripsy source to its driving electronics (e.g., power amplifier and function generator) via the appropriate connectors (e.g., BNC cables) as specified by the manufacturer. 
	NOTE: Ensure that the driving electronics of the histotripsy source can be controlled via the runtime environment used in step 4.1.</li>
	<li>Cover the imaging array with a probe cover and affix the array coaxially in the aperture of the histotripsy source as shown in Figure 2. Be sure to understand the orientation of the imaging plane relative to the orientation of the histotripsy source.</li>
	<li>Connect the imaging array to an ultrasound scanning system. Ensure that this system can control the operation and triggering of the imaging array, and collect imaging data, as per the commands provided by the manufacturer of the scanner.</li>
	<li>Submerge the histotripsy source/imaging array into the tank while degassing as shown in Figure 1A. Gently remove any air bubbles using a syringe from the surface of the histotripsy source or imaging array. 
	NOTE: Immerse the histotripsy source and imaging array completely in water before operation. Avoid touching the surface of the histotripsy source.</li>
	<li>Acquire B-mode images at a rate of 20 frames per second using the imaging array and the built-in commands of the scanner. Adjust the imaging window to ensure visualization of the focus of the histotripsy source in these real-time images. 
	NOTE: It is assumed that the dimensions of the focal region of the therapeutic source are known.</li>
	<li>Set the operating parameters of the histotripsy source, including fundamental frequency (e.g., 1.5 MHz), pulse repetition frequency (e.g., 20-100 Hz), pulse duration (e.g., 1-20 cycles per pulse), and total number of pulses per location (e.g., 100-2,000)18,23,24,33. Modify these parameters if sufficient clot lysis is not achieved or if the bubble activity extends beyond the lumen of the model vessel. To set these parameters, use the protocol provided by the manufacturer of the source or use a programming platform that can communicate with the source (Step 4.3).</li>
	<li>Using the manufacturer&#39;s protocol or programming platform used in step 4.8, run the histotripsy source at the set parameters in degassed water only, without any obstruction in the surrounding environment. Increase the voltage applied to the histotripsy source until a bubble cloud is formed.</li>
	<li>Using the real-time imaging of step 4.7, adjust the position of the imaging array inside the confocal transducer opening until the bubble cloud is located approximately at the center of the image window. The bubble cloud is the region of hyperechoic pixels in the imaging plane (Figure 3). Tighten the screws to hold the imaging array firmly in the transducer opening. 
	NOTE: If the array is aligned properly, the azimuthal position of the bubble cloud should be approximately at 0 mm in the imaging plane. The imaging array may project slightly from the inner surface of the therapy source, and therefore the range position of the bubble cloud may differ from the focal length of the source.</li>
	<li>Identify the bubble cloud location in the imaging plane. Assign the focus of the histotripsy source as the center of the bubble cloud (Figure 3).</li>
	<li>Record the detected focal location (step 4.11) in the imaging window (Figure 3). A possible way to mark the focal position is placing a cursor to note the location in the imaging window, if available with the imaging platform.</li>
	<li>Discontinue insonation and set the voltage applied to the histotripsy source to 0 V.</li>
</ol>
5. Clot preparation
<ol>
	<li>Remove the clot from the pipette by cutting the sealed end with pliers. Let the clot slide into a Petri dish along with the serum. If the clot does not dislodge, gently apply pressure from the other end of the pipette via saline flush to remove the clot.</li>
	<li>Cut the clot to 1 cm length using a scalpel, aiming for a uniform piece from the center (i.e., away from sections of the clot formed at the top or bottom of the pipette).</li>
	<li>Use a cleaning wipe to blot the cut section of the clot gently to remove excess fluid.</li>
	<li>Using tweezers, place the clot section gently on a weighing scale and record the weight.</li>
	<li>Manually raise the flow channel out of the water tank and remove the model vessel from the flow channel.</li>
	<li>Place the clot in the model vessel using tweezers and attach the model vessel again to the flow channel. 
	NOTE: A nylon rod can be placed within the model vessel to prevent the clot from moving downstream due to the flow.</li>
	<li>Lower the flow channel into the tank in such a way that the proximal end of the stage relative to the reservoir is low compared to the distal side. The angling of the stage in this manner prevents trapping of bubbles in the model vessel when plasma is drawn through the flow channel in step 6.1.</li>
	<li>Add 30 mL of plasma to the reservoir using a pipette and monitor the temperature until it reaches at least 36 &#176;C.</li>
	<li>Use a pipette to dispense the rt-PA (80.4 &#181;g in 30 mL of plasma, 2.68 &#181;g/mL) into the plasma reservoir. Stir the plasma with the pipette to ensure a uniform rt-PA distribution within the reservoir.</li>
</ol>
6. Priming the flow channel
<ol>
	<li>Draw plasma into the flow channel from the reservoir via the syringe pump until the plasma fills the model vessel. 
	NOTE: If the clot is not flush with the nylon rod, use short pump draws at 60 mL/min to try to force the plasma downstream the clot or manually draw through the syringe. Limit the amount of plasma drawn in this process or refill the reservoir using additional plasma/rt-PA to ensure 30 mL of solution in the flow channel.</li>
	<li>Using the motorized positioners, align the imaging array parallel to the length of the clot using the imaging script (step 4.7). The parallel alignment enables the user to visually ensure proper placement of the clot and absence of bubbles inside the model vessel.</li>
	<li>Level the model vessel manually and visually ensure that no air bubbles are present using the imaging window (step 4.7).</li>
</ol>
7. Experiment procedure
<ol>
	<li>Pre-treatment 
	&#8203;NOTE: This step is to plan a path for the histotripsy source/imaging array for uniform histotripsy exposure along clot length.
	<ol>
		<li>Align the imaging array using the motorized positioners such that the imaging plane is parallel to the cross-section of the clot (i.e., perpendicular to the orientation described in step 6.2).</li>
		<li>Under guidance via the imaging window (step 4.7), move the histotripsy source to the proximal end of the clot relative to the reservoir using the motorized positioners. At this point, adjust the histotripsy source position such that the marked focal point in step 4.12 aligns with the center of the clot.</li>
		<li>Determine the insonation path along the clot length. To define this path, set three waypoints along the length of the clot (i.e., positions of the motors where histotripsy bubble activity is contained within the clot) in 5 mm increments. Align the waypoints such that the overall motion of the histotripsy source along the path is antegrade with flow in the system (i.e., the first waypoint is at the most proximal end of the clot relative to the reservoir, and the third waypoint is in the distal position relative to the reservoir).</li>
		<li>Prior to finalizing each waypoint, fire test pulses from the histotripsy source with the same insonation parameters as step 4.8 but reduce the overall exposure to 10 total pulses. Adjust the position of the histotripsy source using the motorized positioners if necessary, to contain bubble activity within the clot.</li>
		<li>At each waypoint, save the motor positions using the commands provided by the manufacturer, similar to the step 4.1.</li>
	</ol>
	</li>
	<li>Treatment 
	&#8203;NOTE: This step defines the procedure to treat the clot along its length according to the path defined in the pre-treatment step.
	<ol>
		<li>Run the syringe pump at 0.65 mL/min and wait for meniscus of the plasma to move. This flow rate mimics a near total occlusion of the iliofemoral vasculature24,34.</li>
		<li>Interpolate the path created in step 7.1.3 with intermediate steps between the established waypoints with a fixed step size (e.g., 0.5 mm). The step size is chosen to be smaller than half the width of the focal region as measured along the clot length (elevational direction of the imaging array). Move the histotripsy source using motorized positioners at each path location with insonation parameters defined in step 4.8.</li>
		<li>Monitor/image bubble activity during application of the histotripsy pulse at each path location using the imaging window (step 4.7). Center the image on the histotripsy focus with the image dimensions covering 15 mm in the azimuth and range. Prior to the application of histotripsy pulse at each location, acquire a B-mode image to provide visualization of the clot and model vessel, by creating a script in a programming platform. Ensure the script establishes communication with the scanner using the manufacturer&#39;s commands.</li>
		<li>During the application of the histotripsy pulse, implement the acquisition of acoustic emissions in the script in step 7.2.3 to form passive cavitation images post hoc35. Acquire one passive cavitation image after every 10 treatment pulses. Apply a flat time gain compensation of 50 at 8 incremental depths till the end of the imaging depth. Choose an appropriate acquisition window size such that the entire signal from the clot is captured with minimum loss of energy due to windowing35.</li>
		<li>If off-target bubble clouds are present, adjust the transducer position in situ with the motorized positioners. 
		&#8203;NOTE: Monitor for missed triggers of the imaging array. Adjust the number of acquired imaging data sets to ensure the storage of data is completed before subsequent triggering.</li>
	</ol>
	</li>
</ol>
8. Post experiment procedure
<ol>
	<li>Manually raise the model vessel out of the water tank to drain the perfusate via gravity. Be sure to keep the flow channel levelled to prevent the clot from moving downstream and out of the model vessel during draining.</li>
	<li>Collect the entire perfusate for further analysis in a small beaker (Figure 4A) by drawing plasma solution from the flow channel through the syringe pump at a very low flow rate.</li>
	<li>Disconnect the model vessel and remove the clot. If necessary, inject a small amount of saline into the model vessel to gently dislodge the clot.</li>
	<li>Wipe the clot similar to step 1.2.3. Weigh the clot on the weighing scale for assessing the clot mass loss.</li>
	<li>To analyze the D-dimer content, add 100 mg of aminocaproic acid to a microcentrifuge tube followed by 1 mL of perfusate, and mix well using a pipette. Perform a latex immune-turbidimetric assay to quantify the D-dimer within the sample36.</li>
	<li>To assess hemolysis, add 1 mL of perfusate to centrifuge tubes and spin at 610 x g (3,500 rpm) for 10 min. Combine 0.5 mL of supernatant (concentrate) with 0.5 mL of Drabkins solution and let the mixture rest at room temperature for 15 min. Transfer 200 &#181;L to well plates, as shown in Figure 4B. Use a plate reader to read absorbance at 540 nm, Figure 4C (Drabkins assay37).</li>
	<li>Histology assessment
	<ol>
		<li>Cut a section from the center of the clot of about 2-3 mm in length with a scalpel.</li>
		<li>Add the section to a cassette. Maintain orientation of the section relative to the direction of histotripsy pulse propagation.</li>
		<li>Add 2 mL of low gelling agarose solution prepared in step 3.2.5 into the cassette to fix the clot in place.</li>
		<li>Fix the sample in 10% formalin for 24 h. Place the sample in 70% alcohol after 24 h and perform standard hemotoxylin-eosin staining38.</li>
	</ol>
	</li>
</ol>
9. Passive cavitation image analysis
<ol>
	<li>Process the signals acquired from the imaging array during the histotripsy excitation (step 7.2.4) using an algorithm based on the robust Capon beamformer39 to create an image of acoustic emissions generated by the bubble cloud at each treatment location. 
	NOTE: To generate quantitative images, follow the steps described in Haworth et al.35. Otherwise, each pixel value in the image should represent the relative bubble cloud acoustic energy (units of V2) at each corresponding location.</li>
	<li>Segment the B-mode image aquired in steps 7.2.3 to distinguish between the pixels representing clot and the model vessel.</li>
	<li>Co-register the passive cavitation image with the B-mode image as shown in Figure 5B. Sum up the acoustic energy within the clot over the exposure period35.</li>
</ol>

The authors have nothing to disclose.

The proposed protocol presents a model to quantify treatment efficacy of lysotripsy. While the key details have been discussed, there are certain critical aspects to consider for the success of this protocol. The enzymatic activity of rt-PA has an Arrhenius temperature dependence30. Temperature is also a contributing factor to the speed of sound in water and tissue, and variations in temperature can cause minor alterations of the focal zone geometry. Thus, the water temperature should be carefully...

Thrombosis is the condition of clot formation in an otherwise healthy blood vessel that obstructs circulation1,2. Venous thromboembolism has an annual healthcare cost of $7-10 billion, with 375,000-425,000 cases in the United States3. Pulmonary embolism is the obstruction of the pulmonary artery and is the most serious consequence of venous thromboembolism. The primary source of pulmonary obstruction is deep vein thrombi, primarily from iliofemoral venous segments4,5,6. Deep vein thrombosis (DVT) has inherent sequela besides pulmonary obstructions, with long term complications that result in pain, swelling, leg ulcerations, and limb amputations7,8,9. For critical obstructions, catheter directed thrombolytics (CDT) are the frontline approach for vessel recanalization10. The outcome of CDT depends on a number of factors, including thrombus age, location, size, composition, etiology, and patient risk category11. Moreover, CDT is associated with vascular damage, infections, bleeding complications, and long treatment time10. Next generation devices aim to combine mechanical thrombectomy with thrombolytics (i.e., pharmacomechanical thrombectomy)12,13. Use of these devices lower the lytic dosage leading to reduced bleeding complications, and shortened treatment time12,13,14 as compared to CDT. These devices still retain issues of hemorrhagic side-effects and incomplete removal of chronic thrombi15. An adjuvant strategy is thus needed that can remove the thrombus completely with lower bleeding complications.
One potential approach is histotripsy-aided thrombolytic treatment, referred to as lysotripsy. Histotripsy is a non-invasive treatment modality that uses focused ultrasound to nucleate bubble clouds in tissues16. Bubble activity is generated not via exogenous nuclei, but by the application of ultrasound pulses with sufficient tension to activate nuclei intrinsic to tissues, including clot17,18. The mechanical oscillation of the bubble cloud imparts strain to the clot, disintegrating the structure into acellular debris19. Histotripsy bubble activity provides effective degradation of retracted and unretracted blood clots both in vivo and in vitro20,21,22. Prior studies have23,24 demonstrated that the combination of histotripsy and the lytic&#160;recombinant tissue-type plasminogen activator (rt-PA)&#160;significantly increases treatment efficacy compared to lytic alone or histotripsy alone. It is hypothesized that two primary mechanisms associated with histotripsy bubble activity are responsible for the improved treatment efficacy: 1) increased fibrinolysis due to enhanced lytic delivery, and 2) hemolysis of red blood cells within the clot. The bulk of the clot mass is comprised of red blood cells24, and, therefore, tracking erythrocyte degradation is a good surrogate for ablation of the sample. Other formed clot elements are also likely disintegrated under histotripsy bubble activity but are not considered in this protocol.
Here, a benchtop approach to treat DVT in vitro with lysotripsy is outlined. The protocol describes critical operating parameters of the histotripsy source, assessment of treatment efficacy, and image guidance. The protocol includes designing a flow channel to mimic an iliofemoral venous segment and manufacturing human whole blood clots. The experimental procedure outlines the positioning of the histotripsy source and imaging array to achieve histotripsy exposure along the clot placed in the flow channel. Relevant insonation parameters to attain clot disruption and minimize off-target bubble activity are defined. The use of ultrasound imaging for guidance and assessment of bubble activity is illustrated24. Metrics to quantify treatment efficacy such as clot mass loss, D-dimer (fibrinolysis), and hemoglobin (hemolysis) are outlined23,24,25,26,27. Overall, the study provides an effective means for executing and assessing the efficacy of lysotripsy to treat DVT.

<table><tbody><tr><td>Absorbing sheets</td><td>Precision acoustics</td><td>F28-SMALL-M</td><td>300mm x 300 mm x 10 mm</td></tr><tr><td>Borosilicate Pasteur pippettes</td><td>Fisher Scientific</td><td>1367820A</td><td>14.6 cm length, 2 mL capacity</td></tr><tr><td>Centrifuge tubes</td><td>Eppendorf</td><td>22364111</td><td>1.5 mL capacity</td></tr><tr><td>Drabkin&#039;s assay</td><td>Sigma Aldrich</td><td>D5941-6VL</td><td></td></tr><tr><td>Draw syringe</td><td>Cole-Parmer</td><td>EW-07945-43</td><td>60 mL capacity</td></tr><tr><td>Filter bags</td><td>McMaster-Carr</td><td>5162K111</td><td>Remove particle size upto 1 microns</td></tr><tr><td>Flow channel tubing</td><td>McMaster-Carr</td><td>5154K25</td><td>Polyethylene-lined EVA plastic tubing (Outer diameter: 3/8&quot;, Inner diameter: 1/4&quot;</td></tr><tr><td>Heating elements</td><td>Won Brothers</td><td>HT 300 Titanium</td><td>Titanium rods placed at the bottom of tank</td></tr><tr><td>Imaging array</td><td>Verasonics</td><td>L11-5v</td><td>128 element with sensitivity from -55 to -49 dB</td></tr><tr><td>Low gelling agarose</td><td>Millipore Sigma</td><td>A9414</td><td></td></tr><tr><td>Model vessel</td><td>McMaster-Carr</td><td>5234K98</td><td>6.6 cm length, 0.6 cm inner diameter, 1 mm thickness</td></tr><tr><td>Nanopure water</td><td>Barnstead</td><td>Nanopure Diamond</td><td>ASTM type I, 18 Mohm-cm resistivity</td></tr><tr><td>Plasma</td><td>Vitalant</td><td>4PF000</td><td>Plasma frozen within 24 hours</td></tr><tr><td>Plate reader</td><td>Biotek</td><td>Synergy Neo HST Plate Reader</td><td>For haemoglobin quantification</td></tr><tr><td>Probe cover</td><td>Civco</td><td>610-362</td><td></td></tr><tr><td>Programming platform</td><td>MATLAB (the Mathworks, Natick, MA, USA)</td><td></td><td></td></tr><tr><td>Recombinant tissue-type plasminogen activator (rt-PA)</td><td>Genentech</td><td>Activase</td><td></td></tr><tr><td>Reservoir</td><td>Cole-Parmer</td><td>EW-07945-43</td><td>60 mL capacity</td></tr><tr><td>Syringe pump</td><td>Cole-Parmer</td><td>EW-74900-20</td><td>pump attached to the syringe to draw the flow in the flow channel at a pre-determined fized rate</td></tr><tr><td>Transducer</td><td>In-house customized</td><td></td><td>Eight-element, elliptically-focused transducer (9 cm major axis, 7 cm minor axis and 6 cm focal length), powered by custom designed and built class D amplifier and matching network</td></tr><tr><td>Ultrasound scaning system</td><td>Verasonics</td><td></td><td>Vantage Research Ultrasound System</td></tr><tr><td>Water tank</td><td>Advanced acrylics</td><td>C133</td><td>14 x 14 x 12, 1/2&quot;</td></tr></tbody></table>

an in vitro system to gauge the thrombolytic efficacy of histotripsy and a lytic drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

The protocol outlined in this study highlights the details of venous clot modeling, lysotripsy for clot disruption, and ultrasound imaging in an in vitro setup of DVT. The adopted procedure demonstrates the steps necessary to assess clot disruption due to the combined effects of rt-PA and histotripsy bubble cloud activity. The benchtop setup was designed to mimic the characteristics of a venous iliofemoral vein. Figure 1A shows a model vessel that has the acoustic, mechanical, and geometrica...

Watch this Scientific Journal Video about An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug at JoVE.com

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is ...

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2.7K Views.  University of Chicago. Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

Video: An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Medicine

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

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A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of cerebral blood clots is important for diagnosis, treatment, and prognosis. However, the current approaches to studying the cell components of the clots are mainly based on in situ staining, which is unsuitable for the comprehensive study of the cell components because cells are tightly wrapped in the clots. Previous studies have successfully isolated a fibrinolytic enzyme (sFE) from Sipunculus nudus, which can degrade the cross-linked fibrin directly, releasing the cell components. This study established a comprehensive method based on the sFE to study the cell components of cerebral thrombus. This protocol includes clot dissolving, cell releasing, cell staining, and routine blood examination. According to this method, the cell components could be studied quantitatively and qualitatively. The representative results of experiments using this method are shown.

Author Spotlight: A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots 

This study describes a fast and effective method for the cell component analysis of cerebral blood clots through clot dissolving, cell staining, and routine blood examination.

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...

Biochemistry

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Immunology and Infection

Combining the Chandler Loop and RT-FluFF for Ex Vivo Clot Lysis Monitoring

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates.
The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

Author Spotlight: Advancing Thrombolytic Testing by Integrating Flow Dynamics in In Vitro Models

In-vitro thrombolysis assays have often struggled to replicate in-vivo conditions whether in the model thrombus being digested or in the environment in which thrombolysis is occurring. Herein, we explore how coupling the Chandler loop and Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) is used for high-fidelity, ex-vivo, clot lysis monitoring.

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Neuroscience

Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or&#160;thrombosis. Therefore, characterizing fibrin clot formation is beneficial to the study of thrombosis. Turbidity and thromboelastography (TEG) are both widely utilized in vitro assays for monitoring clot formation. Turbidity dynamically measures the light transmittance through a fibrin clot structure via a spectrometer and is often used in research laboratories. TEG is a specialized viscoelastic technique that directly measures blood clot strength and is primarily utilized in clinical settings to assess patients&#39; hemostasis. With the help of these two tools, this study describes a method for characterizing an in vitro fibrin clot using a simplified fibrinogen/thrombin clot model. Data trends across both techniques were compared under various clotting conditions. Human and bovine fibrin clots were formed side-by-side in this study as bovine clotting factors are often used as substitutes to human clotting factors in clinical and research settings. Results demonstrate that TEG and turbidity track clot formation via two distinct methods and when utilized together provide complementary clot strength and fiber structural information across diverse clotting conditions.

Fibrin is responsible for clot formation during hemostasis and thrombosis. Turbidity assays and thromboelastograhy (TEG) can be utilized as synergistic tools that provide complementary assessment of a clot. These two techniques together can give more insight into how clotting conditions affect fibrin clot formation.

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or&#160;thrombosis. Therefore, ...

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 &#176;C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Summary

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Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices