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 adaptor which allows the animal head rotation. Prepare the anesthetics Ketamine (60 mg/kg)/ Xylazine (10 mg/kg).</li>
	<li>Sterilize the surgical tools including scissors, forceps, micro-needle holders, hemostats, cotton swabs and sutures with autoclave (121 &#176;C at 15 psi for 60 min). Prepare tissue glue and eye ointment. Prepare the 532 nm laser protection goggle for surgeons. 
	NOTE: This protocol describes a major survival surgery procedure and should be conducted using aseptic techniques.</li>
	<li>Set up the illumination system with a 532 nm laser source. Prepare a dental drill.</li>
	<li>Prepare the Rose Bengal solution in saline (10 mg/mL). Place an aliquot bovine thrombin (0.1 U/&#181;L) on ice-bucket.</li>
	<li>Inject Ketoprofen&#160;(4.0 mg/kg) subcutaneously to the mouse as analgesia at 30 min before surgery or use the analgesic regimen recommended by the local institutional guidelines.</li>
</ol>
2. Ligation of the ipsilateral common carotid artery
<ol>
	<li>Anesthetize 10-14 week-old male C57BL/6NCrl mice weighing 22 to 30 g by intramuscular injection of Ketamine (60 mg/kg) and Xylazine (10 mg/kg). 
	NOTE: The entire surgical procedure, encompassing the ligation of the ipsilateral common carotid artery through the monitoring of cerebral blood flow, is expected to take ~120 min. The anesthetic regimen will be typically effective for this entire duration, but the anesthetic depth should be reassessed at least every 15 min. While learning these procedures, it may be necessary to re-dose the anesthesia.</li>
	<li>Perform a toe-pinch to ensure the animal is fully anesthetized. Remove the hair on the left neck for CCA ligation and the head for skull thinning with the hair removal cream.</li>
	<li>Place the mouse on the small animal adaptor in the supine position. Sterilize the surgical area by wiping skin with three alternating swipes of povidone-iodine and 70% ethanol.</li>
	<li>Secure the mouse head using ear bars. Under a dissecting microscope, make a 0.5 cm left-cervical incision using a pair of micro-scissors and straight forceps at about 0.2 cm lateral to the midline.</li>
	<li>Use a pair of fine serrated forceps to pull apart the soft tissue and fascia to expose the left common carotid artery (LCCA). Carefully separate the left CCA from the vagal nerve using a pair of fine smooth forceps.</li>
	<li>Place a permanent double-knot suture around the LCCA using 5-0 silk suture cut into 20 mm segments, and then close the wound using sterile wound clips.</li>
</ol>
3. Skull thinning above the MCA branch and photoactivation
<ol>
	<li>Flip the mouse to prone position on the small animal adaptor. Rotate the nose-clip roll for 15&#176;. Sterilize the surgical area by wiping skin with three alternating swipes of betadine and 70% ethanol.</li>
	<li>Make a 0.8 cm incision in the scalp using a pair of micro-scissors and straight forceps along the left eye and ear to expose the temporalis muscle, which is located between the eye and the ear (Figure 1B).</li>
	<li>Under the dissecting microscope, make a 0.5 cm incision along the edge of temporalis muscle on left parietal bone by a pair of fine serrated forceps. Make a second 0.3 cm vertical incision on temporalis muscle by a micro scissors. Retract the temporal muscle to expose the edge of parietal bone and squamosal bone. Make sure to visualize the landmark of coronal suture between the frontal and the parietal bones (Figure 1B,C).</li>
	<li>Moisture the skull by applying sterile saline to reveal the left MCA. Mark the proximal MCA branch on the squamosal bone with a marker pen. Gently draw a circle for about 1 mm in diameter surrounding the marked area with the pneumatic dental drill (burr speed setting at 50% of speed controller), and then thin the skull about 0.2 mm in depth without touching the underneath dura. Stop the drilling until a very thin layer of bone is left.</li>
	<li>Mix the thrombin (T, 0.1 U/&#956;L, 80 U/kg) and Rose bengal (RB, 10 mg/mL, 50 mg/kg) solution based on the mouse&#8217;s body weight. For example, for a mouse of 25 g body weight, mix 20 &#956;L of thrombin (0.1 U/&#956;L) and 125 &#956;L of RB (10 mg/mL).</li>
	<li>Slowly inject T+RB solution (145 &#181;L per 25 g body weight) into the retro-orbital sinus with an insulin syringe (#31G needle). 
	NOTE: In pilot experiments, the mortality rate of increasing doses of thrombin mixed with the standard dose of RB dye (50 mg/kg) was examined for photoactivation. The mortality was 0% for 80 U/kg thrombin (n=13), 43% for 120 U/kg thrombin (n=7), and 100% for both 160 U/kg (n=5) and 200 U/kg thrombin (n=5). A dose of 80 U/kg thrombin was therefore chosen for this model. Laser speckle contract imaging was also used to exclude the possibility of rampant blood clotting near the orbital cavity after retro-orbital sinus injection of T+RB (Supplementary Figure 1), as well as, widespread fibrin deposition in the contralateral hemisphere that was not subjected to laser illumination (Supplementary Figure 2).</li>
	<li>Apply eye ointment on both eyes to prevent dryness.</li>
	<li>Apply the illuminator with a 532 nm laser light (with 0.5 mW energy) on the drilled site with 2-inch distance for 20 min. Visualize the illumination on the proximal branch of MCA through a laser protection goggle (Figure 1C,D). 
	NOTE: The MCA with 532 nm illumination shows red fluorescence under the goggle. The distal MCA will disappear after 10 min illumination. Exclude the animal if the distal MCA flow is still present after 20 min illumination.</li>
	<li>Stop the laser illumination after 20 min. Close the wound with sterile wound clips.</li>
</ol>
4. Intravital imaging (optional)
NOTE: To characterize the thrombus formation in-vivo, use intraviral imaging by a spin-disk confocal with photoactivation system23.
<ol>
	<li>Make a cranial window ~3 mm in diameter on the parietal bone of skull.</li>
	<li>Place a coverglass on the cranial window and locate the distal MCA (~50 &#956;m in diameter) under a 20x water-immersion objective.</li>
	<li>Label the circulating platelet by tail vein injection of DyLight488-conjugated anti-GPIb&#946; antibody (0.1 mg/kg) at 5 min before imaging.</li>
	<li>Inject the mixture solution of thrombin (80 U/kg) and Rose bengal (50 mg/kg) by retro-orbital at 5 min before imaging.</li>
	<li>Photoactivate the MCA using a 561 nm laser system with laser beam 10 &#956;m in diameter and record the image until the thrombus formation.</li>
</ol>
5. tPA administration
<ol>
	<li>Place the anesthetized animal on a 37 &#176;C warm pad. At the selected post-photoactivation time-point, wet a gauze with ~45 &#176;C warm water and wrap it on the tail for 1 min.</li>
	<li>Inject recombinant human tPA (10 mg/kg) through the tail vein with a 50% bolus and 50% over 30 min by infusion pump. 
	NOTE: Although the clinical dose of recombinant human tPA for acute ischemic stroke treatment is 0.9 mg/kg, a higher dose (10 mg/kg) is commonly used in rodents to compensate for reduced cross-species tPA reactivity. We also followed the standard protocol of tPA-administration in preclinical stroke models, using 50% as a bolus and 50% infused through the tail vein over 30 min.24</li>
</ol>
6. Monitor of cerebral blood flow (CBF)
NOTE: To confirm CBF recovery after tPA treatment, use a two-dimensional laser speckle contrast imaging system15 and record immediately after photothrombosis (step 3.9) or at 24 h after tPA treatment.
<ol>
	<li>Place anesthetized animal in the prone position and make a midline incision on the scalp with the skull exposed.</li>
	<li>Moisturize the skull with sterile saline and gently apply the ultrasound gel on the skull. Avoid any hair and bubble in the gel, which will interfere the CBF signal.</li>
	<li>Monitor CBF in both cerebral hemispheres under laser speckle contrast imager for 10 min.</li>
	<li>After recording the CBF image, close the scalp with tissue glue and return the animal to the cage.</li>
	<li>Analyze CBF in the selected regions and calculate the CBF recovery percentage compared to contralateral region.</li>
	<li>Then, place the animal back to a warm cage for recovery. Monitor the mice for 5-10 min until they recover from anesthesia. Place wetted food in the cage and return it to the animal care facility. 
	NOTE: Provide post-op analgesia as recommended by the local institutional guidelines.</li>
</ol>
7. Infarct volume measurement by triphenyl tetrazolium chloride (TTC) staining
<ol>
	<li>Twenty-four hours after photothrombosis, deeply anesthetize the animal per the local institutional guidelines for nonsurvival surgery. 
	NOTE: We administer tribromoethanol (avertin) 250 mg/kg via intraperitoneal (IP) injection.</li>
	<li>Perform transcardial perfusion with PBS, collect fresh brain and embed in 3% agar gel.</li>
	<li>Section the brain slice with 1 mm thickness by vibratome and incubate in 2% TTC solution for 10 min.</li>
	<li>Quantify the total infarct volume from 6 brain slices as the absolute volume by ImageJ software. 
	NOTE: Brain edema was not used as an outcome measurement for two reasons. First, the TTC stain measures tissue viability (via the mitochondrial reduction activity) which is a more severe consequence than edema. Second, as infarction proceeds, both vasogenic and cytotoxic edema occur and cannot be easily distinguished by the standard brain edema measurement methods. However, we have used anti-immunoglobin (IgG) labeling to assess the integrity of blood-brain-barrier (BBB), and found comparable IgG-extravasation at 6 h after photoactivation in both RB and T+RB stroke models (Supplementary Figure 3).</li>
</ol>
8. Thrombus formation measurement
NOTE: To measure the thrombus formation, collect the brain at 1 h and 2 h after photothrombosis for thrombus measurement in MCA by immunochemistry (IHC) and for fibrin measurement in brain hemisphere by immunoblot, respectively.
<ol>
	<li>Perform the IHC for the characterization of clot composition. Fix the brain with 4% paraformaldehyde overnight and then dehydrate the brain with 30% sucrose for the OCT embedding.</li>
	<li>Section the brain with sagittal orientation in 20 &#956;m thickness, and perform the IHC with specific antibodies against fibrinogen, platelet (glycoprotein IIb), red blood cell (TER119) and blood vessel (isolectin GS-IB4).</li>
	<li>Perform the measurement of fibrin in brain hemisphere by immunoblot with an antibody against fibrinogen.</li>
</ol>

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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>Ketoprofen</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...

Watch this Scientific Journal Video about A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model at JoVE.com

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 ...

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Research

JoVE Journal

Neuroscience

2.6K Views.  National Yang-Ming University School of Medicine. 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.

Video: A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Medicine

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

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 ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

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 ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes and are defined as the sudden blockage of a vessel supplying blood to the brain. Within minutes of the blockage, cells begin to die and result in irreparable tissue damage. Current therapeutic treatments focus on clot removal or lysis to allow for the reperfusion and prevent more severe brain damage. Although transient brain plasticity may salvage some of the damaged tissue over time, significant fractions of patients are left with neurological deficits that will never resolve. There is a lack of therapeutic options to treat neurological deficits caused by stroke, emphasizing the need to develop new strategies to treat this growing patient population. Injectable biomaterials are currently being designed to enhance brain plasticity and improve endogenous repair through the delivery of active agents or stem cells. One method to test these approaches is to utilize a rodent stroke model, inject the biomaterial into the stroke core, and assess repair. Knowing the precise location of the stroke core is imperative for the accurate treatment after stroke, therefore, a stroke model that results in a predictable stroke location is preferable to avoid the need for imaging prior to injection. The following protocol will cover how to induce a photothrombotic stroke, how to inject a hydrogel in a controlled and precise manner, and how to extract and cryosection the brain while keeping the biomaterial intact. In addition, we will highlight how these same hydrogel materials can be used for the co-delivery of stem cells. This protocol can be generalized to the use of other injectable biomaterials into the stroke core.

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes ...