A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

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.