In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

In this procedure, a new near infrared fluorescence catheter or NIRF catheter is used to image two dimensional intra arterial vascular biology and monitor biological processes including atherosclerosis, inflammation, and angiogenesis. First, an experimental model of atherosclerosis in the aorta and iliac of a New Zealand white rabbit is generated next at the appropriate timing. An NIRF Activat Nanosensor is injected to label an inflamed plaque.

24 hours later, a coregistered arterial angiography, intravascular ultrasound, and in vivo and ex vivo intravascular near infrared fluorescence. Images of rabbit, a Roma are required. The aorta and iliac arteries are then dissected out to perform ex vivo fluorescence reflectance imaging, immunohistochemical analysis of the inflamed plaque.

The final results show quantifiable signals that correlate to an inflammatory atherosclerotic plaque. This method can help answer key questions in the field of molecular imaging, such as in vivo, realtime detection of inflammation and plaques, key markers of vulnerable plaques, or the precursors of acute myocardial infarction. The implications of this technique extend towards therapy of atherosclerosis because it can allow realtime in vivo detection of inflammation, which can translate into target specific and patient specific therapies that reduce inflammation in human area.

Human coronary atherosclerosis a key to preventing the dreaded acute myocardial infarction. Though this method can provide insight into coronary atherosclerosis, it can also be applied to other systems including peripheralvascular disease, for example, to characterize lesions in the carotid aorta, iliac or femoral arteries, the culprits of peripheral vascular disease. Following the post balloon denudation and establishment of the atherosclerosis model, animals are maintained on a 1%cholesterol diet for four weeks, and at week five, animals are transitioned to a 0.3%cholesterol diet.

Eight weeks following balloon injury and 24 hours prior to imaging, the rabbit is intravenously injected via an ear vein in order to label proli active inflamed plaques using the injectable nanosensor pro sense 750 VM 110 once 24 hours has passed. Animals are prepared for surgery by shaving the surgical area and then thoroughly disinfecting with 10%povidone iodine animals are then anesthetized and arterial axis is obtained by the right common carotid artery. In order to easily image the tissue intra arterial heparin is administered and a baseline angiography is obtained.

An IVUS catheter is loaded onto a clinical coronary artery 0.014 inch guide wire and inserted into the sheath using fluoroscopic guidance. The radio pack tip of the wire is positioned distally into the right iliac artery. A 100 millimeter pullback spanning the AOR iliac bifurcation to the renal arteries is initiated and images are recorded.

Longitudinal reconstruction of the vessel is obtained and luminal plaque is identified. A 100 millimeter pullback is then used to cover the entire length of the aorta. Longitudinal reconstructions are generated from the series for two dimensional axial images using MATLAB programming software.

The NIRF catheter is loaded onto the wire. The catheter is carefully inserted into the sheath and the imaging head is positioned distally into the left iliac artery while performing multiple automated NIRF catheter pullbacks. The fluorescent signals within zones of atherosclerosis are noted.

These images are recorded and are further processed using MATLAB software, appropriate scaling and windowing based on the range of signal achieved. The animal is then euthanized and aortoiliac tissue is isolated ex-vivo. The atherosclerotic aorta and iliac arteries are identified and dissected free from the surrounding tissues.

In addition, small two times two centimeter pieces of liver, kidney, spleen, and heart are also obtained. Once isolated, the arterial tree is perfused with normal saline until the inferior vena caver is clear of blood. Once Exvivo NIRF has been completed, the dissected AOR iliac tissue is placed in 10 to 20 cubic centimeters of normal saline and transported for fluorescence reflectance imaging or FRI analysis.

The aorta and iliac vessels are elongated to approximate real-time lengths and images are obtained at multiple wavelengths using a commercially available fluorescence reflectance imaging system equipped with multichannel filters, a series of exposure times are utilized for each wavelength and acquired. Images are exported as DICOM files for further analyses as positive and negative controls. Specific organs such as liver, spleen, kidney and heart, are imaged at similar channels and exposure times.

Any areas of increased fluorescent signal in the near infrared channel are noted in atherosclerotic arteries. Increased signal correlates with areas of increased plaque, cathepsin B signal and plaque macrophages, markers of inflammation that demarcate high risk vulnerable plaques. The areas of normal non-injured tissue such as the left iliac artery and the areas of plaque are identified, small five to 10 millimeter rings of tissue are embedded in optimal cutting temperature or OCT media until needed.

For sectioning blocks are stored at minus 80 degrees Celsius using standard techniques for sectioning and immunohistochemical analysis. Hematin and eosin staining and RAM 11 staining are performed. DICOM files are used to process imaging data from NIRF and FRI, which are taken at near infrared.

750 nanometer channel and pullbacks are processed using MATLAB and OIC software respectively. Proper windowing to display full range of signal intensity is achieved. Final images are exported as TIFF files.

Files are imported into standard image analysis. Software and images are aligned based on reference points such as the vertebrae, iliac bifurcation, and renal artery. On the angiogram, areas of normal vessel and plaque are identified.

Regions of interest or ROI are manually traced for normal tissue and areas of plaque to guide appropriate tracing. The longitudinal IVUS image of the vessel is used and identification of normal vessel and plaque are easily identified. Mean signal intensities are acquired using MATLAB and oix respectively for both FRI and NIRF images.

Finally, targeted background or TBR ratios are calculated for plaque zones. To calculate the TBR, the mean ROI signal of plaque area is divided by the ROI signal of background noise. Upon completion of the above protocol, we can identify and characterize areas of augmented kassin or inflammatory molecule protease activity in an inflammatory plaque within the aorta and iliac vessels injection of an activat nanosensor.

The pro sense VM 110 allows us to identify the proteolytic active plaque. These appear as bright or signal intense zones when imaged using FRI in the near infrared channel. The NIRF pullbacks correlate with increased signal intensity by FRI and alignments with IVUS, which allow anatomical registration of NIRF signals calculated plaque tbrs obtained from FRI and NIRF with similar immunohistochemical analysis of bright plaque confirms intense presence of RAM 11 activity in areas of plaque Once mastered, this technique can be done in about two to four hours if it's performed properly.

While attempting this procedure, it is important to remember to be careful in in the consistent windowing and imaging data and proper alignment with angiography. Intravascular ultrasound and FRI are critical to correct interpretation of the data After its development. This technique paved the way for researchers in the field of molecular imaging to explore atherosclerosis in humanized coronary vessels.

After watching this video, you should have a good understanding of how to utilize a multimodal imaging strategy that utilizes this novel in vivo intravascular nerve catheter to image and quantify inflammatory plaque in proteolytic active inflamed rapid atheroma. Don't forget that working with surgical instruments and bodily fluids, such as blood can be hazardous and precautions such as careful surgical techniques and personal safety materials such as gloves and masks should always be taken while performing this procedure.