This work was supported by the NIH grants (NS108763, NS100419, NS095064, and HD080429 to C.Y. K.; and NS106592 to Y.Y.S.).

This protocol is approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Virginia and follows the National Institutes of Health Guideline for Care and Use of Laboratory Animals. Figure 1A outlines the sequence of surgical procedures of this protocol.
1. Surgery setup
<ol>
	<li>Place a warming pad with temperature setting at 37 &#176;C on the small animal adaptor at least 15 minutes before the surgery. Prepare a nose-clip roll for...

<ol>
	<li>Lyden, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thrombolytic Therapy for Acute Stroke. 3/e. , (2015).</li><li>Linfante, I., Cipolla, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+reperfusion+therapies+in+the+era+of+mechanical+thrombectomy.">Improving reperfusion therapies in the era of mechanical thrombectomy.</a> Translational Stroke Research. 7 (4), 294-302 (2016).</li><li>Campbell, B. C., 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=Endovascular+Therapy+for+Ischemic+stroke+with+perfusion-imaging+selection.">Endovascular Therapy for Ischemic stroke with perfusion-imaging selection.</a> The New England Journal of Medicine. 372 (11), 1009-1018 (2015).</li><li>Hossmann, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+pathophysiologies+of+focal+brain+ischemia:+implications+for+translational+stroke+research.">The two pathophysiologies of focal brain ischemia: implications for translational stroke research.</a> Journal of Cerebral Blood Flow and Metabolism. 32 (7), 1310-1316 (2012).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</li><li>Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S., Ginsberg, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+brain+infarction+by+photochemically+initiated+thrombosis.">Induction of reproducible brain infarction by photochemically initiated thrombosis.</a> Annals of Neurology. 17 (5), 497-504 (1985).</li><li>Watson, B. D., Prado, R., Veloso, A., Brunschwig, J. P., Dietrich, W. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothrombotic+stroke+as+a+model+of+ischemic+stroke.">Photothrombotic stroke as a model of ischemic stroke.</a> Translational Stroke Research. 9 (5), 437-451 (2018).</li><li>Karatas, H., 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=Thrombotic+distal+middle+cerebral+artery+occlusion+produced+by+topical+FeCl(3)+application:+a+novel+model+suitable+for+intravital+microscopy+and+thrombolysis+studies.">Thrombotic distal middle cerebral artery occlusion produced by topical FeCl(3) application: a novel model suitable for intravital microscopy and thrombolysis studies.</a> Journal of Cerebral Blood Flow and Metabolism. 31 (3), 1452-1460 (2011).</li><li>Orset, C., 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=Mouse+model+of+in+situ+thromboembolic+stroke+and+reperfusion.">Mouse model of in situ thromboembolic stroke and reperfusion.</a> Stroke. 38 (10), 2771-2778 (2007).</li><li>Orset, C., 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=Efficacy+of+Alteplase+in+a+mouse+model+of+acute+ischemic+stroke:+A+retrospective+pooled+analysis.">Efficacy of Alteplase in a mouse model of acute ischemic stroke: A retrospective pooled analysis.</a> Stroke. 47 (5), 1312-1318 (2016).</li><li>Kudo, M., Aoyama, A., Ichimori, S., Fukunaga, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+animal+model+of+cerebral+infarction.+Homologous+blood+clot+emboli+in+rats.">An animal model of cerebral infarction. 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Y., 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=Sickle+mice+are+sensitive+to+hypoxia/ischemia-induced+stroke+but+respond+to+tissue-type+plasminogen+activator+treatment.">Sickle mice are sensitive to hypoxia/ischemia-induced stroke but respond to tissue-type plasminogen activator treatment.</a> Stroke. 48 (12), 3347-3355 (2017).</li><li>Sun, Y. Y., Kuan, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thrombotic+stroke+model+based+on+transient+cerebral+hypoxia-ischemia.">A thrombotic stroke model based on transient cerebral hypoxia-ischemia.</a> Journal of Visualized Experiments. (102), e52978 (2015).</li><li>Pena-Martinez, C., 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=Pharmacological+modulation+of+neutrophil+extracellular+traps+reverses+thrombotic+stroke+tPA+(tissue-type+plasminogen+activator)+resistance.">Pharmacological modulation of neutrophil extracellular traps reverses thrombotic stroke tPA (tissue-type plasminogen activator) resistance.</a> Stroke. 50 (11), 3228-3237 (2019).</li><li>Denorme, F., 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=ADAMTS13-mediated+thrombolysis+of+t-PA-resistant+occlusions+in+ischemic+stroke+in+mice.">ADAMTS13-mediated thrombolysis of t-PA-resistant occlusions in ischemic stroke in mice.</a> Blood. 127 (19), 2337-2345 (2016).</li><li>Marder, V. J., 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=Analysis+of+thrombi+retrieved+from+cerebral+arteries+of+patients+with+acute+ischemic+stroke.">Analysis of thrombi retrieved from cerebral arteries of patients with acute ischemic stroke.</a> Stroke. 37 (8), 2086-2093 (2006).</li><li>Bacigaluppi, M., Semerano, A., Gullotta, G. S., Strambo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+from+thrombi+retrieved+in+stroke+due+to+large+vessel+occlusion.">Insights from thrombi retrieved in stroke due to large vessel occlusion.</a> Journal of Cerebral Blood Flow and Metabolism. 39 (8), 1433-1451 (2019).</li><li>Sun, Y. Y., 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=A+murine+photothrombotic+stroke+model+with+an+increased+fibrin+content+and+improved+responses+to+tPA-lytic+treatment.">A murine photothrombotic stroke model with an increased fibrin content and improved responses to tPA-lytic treatment.</a> Blood Advances. 4 (7), 1222-1231 (2020).</li><li>Su, E. J., 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=Activation+of+PDGF-CC+by+tissue+plasminogen+activator+impairs+blood-brain+barrier+integrity+during+ischemic+stroke.">Activation of PDGF-CC by tissue plasminogen activator impairs blood-brain barrier integrity during ischemic stroke.</a> Nature Medicine. 14 (7), 731-737 (2008).</li><li>Gupta, A. 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The authors have nothing to disclose.

The traditional RB photothrombotic stroke, introduced in 1985, is an attractive model of focal cerebral ischemia for simple surgical procedures, low mortality, and high reproducibility of brain infarction.5 In this model, the photodynamic dye RB rapidly activates platelets upon light excitation, leading to dense aggregates that occlude the blood vessel5,8,23. However, the small amount of fibrin in RB-indu...

Endovascular thrombectomy and tPA-mediated thrombolysis are the only two U.S. Food and Drug Administration (FDA)-approved therapies of acute ischemic stroke, which afflicts ~700,000 patients annually in the United States1. Because the application of thrombectomy is limited to large vessel occlusion (LVO), while tPA-thrombolysis may alleviate small vessel occlusions, both are valuable therapies of acute ischemic stroke2. Moreover, the combination of both therapies (e.g., initiation of tPA-thrombolysis within 4.5 hours of stroke onset, followed by thrombectomy) improves reperfusion and the functional outcomes3. Thus, optimizing thrombolysis remains an important goal for stroke research, even in the era of thrombectomy.
Thromboembolic models are an essential tool for preclinical stroke research aiming to improve thrombolytic therapies. This is because mechanical vascular occlusion models (e.g., intraluminal suture MCA occlusion) do not produce blood clots, and its fast recovery of cerebral blood flow after the removal of mechanical occlusion is overly idealized4,5. To date, major thromboembolic models include photothrombosis6,7,8, topical ferric chloride (FeCl3) application9, microinjection of thrombin into the MCA branch10,11, injection of ex vivo (micro)emboli into the MCA or common carotid artery (CCA)12,13,14, and transient hypoxia-ischemia (tHI)15,16,17,18. These stroke models differ in the histological composition of ensuing clots and the sensitivity to tPA-mediated lytic therapies (Table 1). They also vary in the surgical requirement of craniotomy (needed for in situ thrombin injection and topical application of FeCl3), the consistency of infarct size and location (e.g., CCA-infusion of microemboli yield very variable outcomes), and global effects on the cardiovascular system (e.g., tHI increases the heart rate and cardiac output to compensate for hypoxia-induced peripheral vasodilation).
The RB dye-based photothrombotic stroke (PTS) model has many attractive features, including simple craniotomy-free surgical procedures, low mortality (typically &#60; 5%), and a predictable size and location of infarct (in the MCA-supplying territory), but it has two major limitations.8 The first caveat is weak-to-nil response to tPA-mediated thrombolytic treatment, which is also a drawback of the FeCl3 model7,19,20. The second caveat of PTS and FeCl3 stroke models is that the ensuing thrombi consist of densely-packed platelet aggregates with a small amount of fibrin, which not only lead to its resilience to tPA-lytic therapy, but also deviates from the pattern of intermixed platelet:fibrin thrombi in acute ischemic stroke patients21,22. In contrast, the in situ thrombin-microinjection model mainly comprises polymerized fibrin and a uncertain content of platelets10.
Given the above reasoning, we hypothesized that admixture of RB and a sub-thrombotic dose of thrombin for MCA-targeted photoactivation through thinned skull may increase the fibrin component in the resultant thrombi and boost the sensitivity to tPA-mediated lytic treatment. We have confirmed this hypothesis,23 and herein we describe detail procedures of the modified (T+RB) photothrombotic stroke model.

<table>
	<tbody>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride (TTC)</td>
			<td>Sigma</td>
			<td>T8877</td>
			<td>infarct</td>
		</tr>
		<tr>
			<td>4-0 Nylon monofilament suture</td>
			<td>LOOK</td>
			<td>766B</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>Harvard Apparatus</td>
			<td>624143</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>543nm laser beam</td>
			<td>Melles Griot</td>
			<td>25-LGP-193-249</td>
			<td>photothrombosis</td>
		</tr>
		<tr>
			<td>adult male mice</td>
			<td>Charles River</td>
			<td>C57BL/6</td>
			<td>10~14 weeks old (22~30 g)</td>
		</tr>
		<tr>
			<td>Anesthesia bar for mouse adaptor</td>
			<td>machine shop, UVA</td>
			<td></td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Avertin (2, 2, 2-Tribromoethanol)</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>euthanasia</td>
		</tr>
		<tr>
			<td>Dental drill</td>
			<td>Dentamerica</td>
			<td>Rotex 782</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Digital microscope</td>
			<td>Dino-Lite</td>
			<td>AM2111</td>
			<td>brain imaging</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus</td>
			<td>SZ40</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Fine curved forceps (serrated)</td>
			<td>FST</td>
			<td>11370-31</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Fine curved forceps (smooth)</td>
			<td>FST</td>
			<td>11373-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>goat anti-rabbit Alexa Fluro 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Halsted-Mosquito hemostats</td>
			<td>FST</td>
			<td>13008-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Heat pump with warming pad</td>
			<td>Gaymar</td>
			<td>TP700</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>infusion pump</td>
			<td>KD Scientific</td>
			<td>200</td>
			<td>thrombolytic treatment</td>
		</tr>
		<tr>
			<td>Insulin syringe with 31G needle</td>
			<td>BD</td>
			<td>328291</td>
			<td>photothrombosis</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>CCM, UVA</td>
			<td></td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Laser protective google 532nm</td>
			<td>Thorlabs</td>
			<td>LG3</td>
			<td>photothrombosis</td>
		</tr>
		<tr>
			<td>Meloxicam SR</td>
			<td>CCM, UVA</td>
			<td></td>
			<td>NSAID analgesia</td>
		</tr>
		<tr>
			<td>micro needle holders</td>
			<td>FST</td>
			<td>12060-01</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>micro scissors</td>
			<td>FST</td>
			<td>15000-03</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>MoorFLPI-2 blood flow imager</td>
			<td>Moor</td>
			<td>780-nm laser source</td>
			<td>Laser Speckle Contrast Imaging</td>
		</tr>
		<tr>
			<td>Mouse adaptor</td>
			<td>RWD</td>
			<td>68014</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Puralube Vet ointment</td>
			<td>Fisher</td>
			<td>NC0138063</td>
			<td>eye dryness prevention</td>
		</tr>
		<tr>
			<td>Retractor tips</td>
			<td>Kent Scientific</td>
			<td>Surgi-5014-2</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Rose Bengal</td>
			<td>Sigma</td>
			<td>198250</td>
			<td>photothrombosis</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T7513</td>
			<td>photothrombosis</td>
		</tr>
		<tr>
			<td>Tissue glue</td>
			<td>Abbott Laboratories</td>
			<td>NC9855218</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>tPA</td>
			<td>Genetech</td>
			<td>Cathflo activase 2mg</td>
			<td>thrombolytic treatment</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Stoelting</td>
			<td>51425</td>
			<td>TTC infacrt</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>CCM, UVA</td>
			<td></td>
			<td>anesthesia</td>
		</tr>
	</tbody>
</table>

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.

First, we compared the fibrin content in RB versus T+RB photothrombosis-induced blood clots. Mice were sacrificed by transcardial perfusion of fixatives at 2 h after photoactivation, and brains were removed for immunofluorescence staining of the MCA branch in longitudinal and transverse planes. In RB photothrombosis, the MCA branch was densely packed with CD41+ platelets and little fibrin (Figure 2A,C). In contrast, the MCA branch in T+RB photothrombosis was occlu...

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A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

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 ...

We want to create a stroke model that produce blood clot that's similar to patient samples and respond to the clinical relevant thrombolytic therapy. Therefore, we modify the classical photothrombotic stroke model and mixing thrombin and the photodynamic dye rose bengal prior to the photoactivation. We are pleased that this procedure indeed creates blood clot that are highly sensitive to tPA-mediated thrombolysis.This modified photothrombotic stroke model use very simple surgical procedures with a low mortality rate produced a highly consistent infarct size and location. You also produce a fibrin and platelet mixed blood clot that are similar to those in the acute stroke patient. Therefore, we think it's quite useful for preclinical stroke research to develop more effective thrombolytic therapies.We hope more people use our model. Demonstrating the procedure will be Dr.Yu-Yo Sun, our research assistant professor in our group. 30 minutes before surgery, prepare the mouse by injecting an analgesic.After anesthetizing the mouse, perform a toe pinch to ensure it is fully anesthetized. Then remove the hair on the left neck and head with hair removal cream. Place the mouse on the small animal adapter in a supine position, and sterilize the surgical skin area by wiping it with three alternating swipes of povidone iodine and 70%ethanol.Under a dissecting microscope, make a 0.5-centimeter left cervical incision using a pair of micro scissors and straight forceps at about 0.2 centimeters lateral to the midline. Next, using a pair of fine serrated forceps, pull apart the soft tissue and fascia to expose the LCCA. Then using a pair of fine smooth forceps, carefully separate the LCCA from the vagal nerve.Place a permanent double-knot suture around the LCCA using a 5-0 silk suture. Flip the mouse to a prone position, and rotate the nose clip roll by 15 degrees. Then sterilize the surgical area by wiping the skin with three alternating swipes of Betadine and 70%ethanol.Make a 0.8-centimeter incision in the scalp using a pair of micro scissors and straight forceps along the left eye and ear to expose the temporalis muscle. Next, make a 0.5-centimeter incision along the edge of the temporalis muscle on the left parietal bone. Then make a second 0.3-centimeter vertical incision on the temporalis muscle and retract the muscle to expose the edge of the parietal bone and squamosal bone.Make sure to visualize the landmark of the coronal suture between the frontal and parietal bones. Next, moisten the skull by applying sterile saline to reveal the left MCA, and mark the proximal MCA branch on the squamosal bone with a marker. Gently draw a one-millimeter-diameter circle surrounding this marked area with a pneumatic dental drill.Then thin the skull about 0.2 millimeters deep without touching the underneath dura. Stop the drilling when a very thin layer of bone remains. Next, mix the thrombin and rose bengal solution based on the mouse's body weight, and slowly inject the mixture into the retro-orbital sinus with a 31-gauge needle.Apply eye ointment on both eyes to prevent dryness. Apply the illuminator with a 532-nanometer laser light on the drilled site within a two-inch distance for 20 minutes. Visualize the illumination at the proximal branch of the MCA through laser projection goggles.After 20 minutes, stop the laser illumination. Make a cranial window of three millimeters in diameter on the parietal bone of the skull, and place a cover glass on it. Then locate the distal MCA under a 20x water immersion objective lens.Label the circulating platelet by tail vein injection of DyLight 488 conjugated anti-GP1b-beta antibody five minutes before imaging. Then retro-orbitally inject the thrombin and rose bengal solution mixture. Photoactivate the MCA using a 532-nanometer laser system with a 10-micrometer-diameter laser beam, and record the image until thrombus formation.For tPA administration, place the anesthetized animal on a 37-degree-Celsius warm pad. Wrap its tail with a 45-degree-Celsius warm wet gauze for one minute at the selected post-photoactivation time point. Next, inject the recombinant human tPA through the tail vein.Inject 50%as a bolus, and infuse the remaining 50%over 30 minutes using an infusion pump. To monitor cerebral blood flow, make a midline incision on the scalp with the skull exposed. Moisturize the skull with sterile saline, and gently apply the ultrasound gel on it, making sure to avoid hair or air bubbles in the gel.Monitor the cerebral blood flow in both cerebral hemispheres under the laser speckle contrast imager for 10 minutes. Immunofluorescence labeling shows that in the rose bengal dye-based photothrombosis, the MCA branch was densely packed with CD41-positive platelets and little fibrin. In contrast, in the thrombin plus rose bengal photothrombosis, the MCA branch was occluded by randomly mixed platelet and fibrin clots.The immunoblotting analysis showed a greater than twofold increase in fibrin deposition in the ipsilateral hemisphere in the thrombin plus rose bengal photothrombosis compared to rose bengal alone. Confocal microscope-based intravital imaging showed that an intravenous thrombin injection failed to induce platelet aggregates, even under laser illumination. Platelets formed homogeneous clots in the rose bengal photothrombosis model but uneven aggregates with multiple faint regions in the thrombin plus rose bengal model.The CBF of the same mouse at pre and 24 hours post tPA versus vehicle treatment was measured by laser speckle contrast imaging and normalized to the contralateral hemisphere. In the rose bengal photothrombosis, the tPA treatment led to a trend of CBF recovery, particularly in the ischemic border area compared to vehicle-treated mice. In the thrombin plus rose bengal photothrombosis, the recovery of CBF in tPA-treated mice was more prominent, and the proximal MCA branches often became visible at 24 hours.In rose bengal photothrombosis, a similar infarct size was detected in vehicle-treated and tPA-treated mice. In contrast, in thrombin plus rose bengal photothrombosis, the tPA lytic treatment significantly reduced infarction at 0.5, one, or two hours, but not at six hours post photoactivation compared to vehicle treated mice, Thrombin-mediated fibrin generation is crucial for thrombus formation in this procedure. This method can be used to investigate clot composition and to further study thrombolytic treatments.Following this procedure, other methods of thrombolysis therapy can be applied and the later efficacy determined. Adding thrombin makes it possible to adjust the clot composition from fibrin poor to fibrin rich, allowing researchers to develop clinically relevant therapeutic strategies.

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Research

JoVE Journal

Neuroscience

2.4K Views.  National Yang-Ming University School of Medicine. Vogliamo creare un modello di ictus che produca coaguli di sangue simili ai campioni dei pazienti e risponda alla terapia trombolitica clinicamente rilevante. Pertanto, modifichiamo il classico modello di ictus fototrombotico e mescoliamo la trombina e il colorante fotodinamico rosa bengala prima della fotoattivazione. Siamo lieti che questa procedura crei effettivamente coaguli di sangue altamente sensibili alla trombolisi mediata da tPA.Questo modello di ictus fototrombotico modifica...