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

The overall goal of this direct thrombus imaging technique is to quantitatively visualize fluorescently labeled cerebral thromboemboli in vivo and ex vivo, using gold nanoparticles, computed tomography, and near-infrared fluorescent imaging. This method can help answer key questions in the hemostasis and thrombosis field, such as time or dose dependent and serious orthopedic response of cerebral thromboemboli to tissue plasminogen activator or new thrombolytic agents under investigation. The main advantage of this technique is, after intravenous injection of gold nanoparticles, high resolution micro-CT enables capturing dynamic evolution of cerebral thromboemboli by visualizing their locations and burden quantitatively in vivo then corroborated by near-infrared fluorescent thrombus imaging ex vivo.

Demonstrating the procedure will be done by Jeong-Yeon Kim, who is a post-doc from my laboratory. After collecting 300 to 1, 000 microliters of arterial blood by cardiac puncture, add 70 microliters of the sample to 30 microliters of the appropriate activated coagulation factor, 13 mediated fibrin cross-linking activity sensitive fluorescent probe, and load a three milliliter syringe, equipped with a 23 gauge needle, with the resulting mixture. Next, inject the labeled blood into a 20 centimeter long polyethylene tube and allow the blood to clot at room temperature.

After two hours, transfer the tube to four degrees Celsius for 22 hours, then cut the tube into 1.5 centimeter pieces. Using a three milliliter syringe filled with PBS, expel the thrombus from each piece of tubing into individual wells of a six well plate containing PBS. Wash the thrombi three times with PBS, then insert a saline filled one milliliter syringe equipped with a 30 gauge needle into one end of 15 centimeter PE10 tube and carefully draw a single washed thrombus into the opposite end of the tube.

Then, connect the thrombus loaded PE10 tube with a three centimeter long PE50 tube with 200 micron diameter tapered end. After confirming anesthesia by a lack of response to toe pinch, disinfect the surgical area with Betadine and 70%alcohol and use a scalpel to make a one centimeter vertical incision between the left ear and eye. Glue the distal end of the optical fiber of a laser Doppler flow meter onto the exposed left temporal bone surface one millimeter left and four millimeters below the bregma and begin the Doppler monitoring.

Then make a three centimeter vertical midline incision through the perivascular soft tissues to expose the left carotid bulb area. Using a sterile 6-0 black silk suture, ligate the left proximal common carotid artery, followed by the ligation of the left internal carotid artery and the left pterygopalatine artery with sterile 7-0 black silk sutures. Using a mono-polar electrical cautery, cauterize the left superior thyroid artery, then use sterile 7-0 black silk sutures to loosely ligate the left proximal external carotid artery and to tightly ligate the distal end of the vessel.

Use micro-scissors to make a 0.2 to 0.25 micron hole between the ligated sites of the external carotid artery and insert the thrombus containing catheter into the hole while loosening the proximal external carotid artery ligation. Advance the catheter into the common carotid artery to cinch the catheter in place and retighten the proximal external carotid artery ligation. Cut the distal end of the external carotid artery by cautery and rotate the free proximal end of the external carotid artery clockwise to align the vessel with the internal carotid artery while withdrawing the catheter.

Then, loosen the internal carotid artery ligature and immediately advance the catheter about nine millimeters into the middle cerebral anterior cerebral artery bifurcation. Now, retighten the internal carotid artery suture around the catheter and gently depress the syringe to inject the thrombus into the bifurcation area. If a blood flow greater than or equal to 30%lower than the baseline is observed on the Doppler, remove the catheter and immediately ligate the proximal end of the external carotid artery.

To image the thrombus by micro-computer tomography at the appropriate experimental time point, dilute fibrin targeted gold nanoparticles to a 10 milligrams per milliliter of 10 millimolar PBS concentration and sonicate the thrombus imaging agent for 30 minutes to ensure homogeneous suspension of the nanoparticles. Then inject 300 microliters of the fibrin-targeted particles into the penile vein of the experimental animal. Transfer the animal to the bed of the micro-CT imager and use a string attached to a pin to gently pull on the upper front teeth to straighten the neck.

Five minutes after the gold nanoparticles have been injected begin the imaging. To visualize the thrombus by ex vivo near-infrared imaging, place the excised brain tissue in the imager with the base pointing up. Obtain images of the fluorescently labeled thromboemboli in the arteries of the circle of Willis.

Then, position the tissue with the vertex pointing up and image the cortical thromboemboli. Hydrate the tissue with a few drops of saline, then using brain matrix device according to the manufacturer's instructions, quickly obtain six two millimeter thick coronal brain slices. Keeping the slices hydrated with saline, obtain additional near-infrared images of both the front and back surfaces of the sections.

To quantify the thrombus area after micro-CT imaging, open the image sequence files and select Image followed by Type followed by 8-bit to convert the images to 8-bit grayscale. When one pixel of CT image files corresponds to 0.053 millimeters, use Analyze Set Scale to enter one, 0.053, and millimeters for Distance in pixels, Known distance, and Unit of length, respectively. Then, under Edit, choose Selection followed by Specify to place a region of interest on an area of the brain parenchyma without a thrombus or bone related hyperdense region.

Because the specified region of interest applies to every slice of the stack, confirm that the region does not overlap with a hyperdense region in any of the slices. Then, under Plugin, select Region of Interest and Background Subtraction from the Region of Interest and enter two for the Number of standard deviations from the mean. Find thromboemboli-related hyperdense lesions and zoom into the lesional area.

Now, choose Image followed by Adjust and Threshold and enter a Lower Threshold Level of 22 and an Upper Threshold Level of 255. Select Over/Under to display the pixels below the lower threshold value in blue, the thresholded pixels in grayscale, and the pixels above the upper threshold value in green. Then, use the Freehand selection tool to draw regions of interest surrounding the thromboemboli-related hyperdense lesions without any bony areas.

Use Analyze, Set Measurements to choose Area, Mean gray value, and Integrated density area times mean. Check the following options, Limit to threshold and Display label. Use Analyze Measure to get the quantified data, then save the results.

These baseline micro-CT images were obtained in vivo five minutes after the injection of fibrin targeted gold nanoparticles at one hour after embolic stroke in mice. The cerebral thrombus can be clearly visualized in the middle cerebral anterior cerebral artery bifurcation area of the distal internal carotid artery. Follow-up micro-CT images demonstrate no changes in the circle of Willis thrombus in the saline treated animals, however tissue plasminogen activator treated animals exhibit a gradual dissolution of the circle of Willis thrombus over time, indicated by the blue arrowheads.

There is a strong correlation between the in vivo CT density and the ex vivo near-infrared imaging for residual thromboemboli at 24 hours. For example, in these images the hyperdense micro-CT areas, visualized by the fibrin targeting gold particles, directly correspond to the near-infrared signal areas identified by the coagulation factor 13A mediated, fibrin cross-linking activity sensitive fluorescent probe that binds to the thrombus during the preformed clot maturation process. Further, as visualized in these images, TTC staining can be used to assess the effects of thrombolysis in ischemic brain damage in control saline compared to tissue plasminogen activator treated animals.

Once mastered, this technique for the in vivo micro-CT imaging and ex vivo near-infrared fluorescent thrombus imaging can be done in about one hour if it is performed properly and when the imaging probes are available. While attempting this procedure, it is important to remember to use non-aggregated and fibrin targeted gold nanoparticles for micro-CT based direct cerebral thrombus imaging. Following this procedure, other methods like electron microscopy or ICPMS, which is inductively coupled plasma mass spectrometry, can be performed in order to answer additional questions like tissue distribution and blood kinetics of gold nanoparticles.

After its development, this technique paved the way for researchers in the field of stroke or cardiovascular research to explore thromboembolism in animal models that were inside to coronary thrombosis or embolic cerebral infraction. After watching this video, you should have a good understanding of how to make fluorescent labeled clots for thromboembolic stroke in mice and perform serial imaging and quantification of cerebral thromboemboli in vivo then ex vivo, thereby monitoring the thrombus evolution.