Radiopaque alginate microcapsules for X-ray visualization and immunoprotection of cellular therapeutics.

Alginate-poly-L-lysine-alginate (APA) microcapsules have been explored as vehicles for therapeutic drug and cell delivery. The permselectivity of these capsules provides a unique means of controlled drug release and immunoisolation of encapsulated cells. Immunoisolation is especially attractive as it abrogates the need for chronic immunosuppressive therapy and opens up the possibility for the delivery of numerous cell sources including xenogeneic grafts. APA microcapsules containing cellular therapeutics have proven effective in the short-term treatment of a wide range of diseases requiring enzyme or endocrine replacement therapy, including type I diabetes. If these microcapsules could be noninvasively monitored with X-ray imaging modalities (i.e., fluoroscopy, CT, and digital subtraction angiography), questions such as the ideal transplantation site, the best means of delivery, and the long-term survival of grafts could be better addressed. We have developed two novel alginate-based radiopaque microcapsule formulations containing either barium sulfate (Ba X-Caps) or bismuth sulfate (Bi X-Caps). As compared to conventional, nonradiopaque APA capsules, Ba X-Caps and Bi X-Caps containing human cadaveric islets resulted in a decrease in cellular viability of less than 5% up to 14 days after encapsulation. Both radiopaque capsules were found to be permeable to lectins < or =75 kDa, but were impermeable to lectins > or =120 kDa, thus ensuring the blockage of the penetration of antibodies while allowing free diffusion of insulin and nutrients. The glucose-responsive insulin secretion of the radiopaque encapsulated human islets was found to be unaltered compared to that of unlabeled controls, with human C-peptide levels ranging from 3.21 to 2.87 (Ba X-Caps) and 3.23 to 2.87 (Bi X-Caps) ng/islet at 7 and 14 days postencapsulation, respectively. Using fluoroscopy, both Ba X-Caps and Bi X-Caps could be readily visualized as single radiopaque entities in vitro. Furthermore, following transplantation in vivo in mice and rabbits, single capsules could be identified with no significant change in contrast for at least 2 weeks. This study represents the first attempt at making radiopaque microcapsules for X-ray guided delivery and imaging of cellular therapeutics. While human cadaveric islets were used as a proof-of-principle, these radiopaque capsules may have wide ranging therapeutic applications for a variety of cell types.

MR-trackable intramyocardial injection catheter.

There is growing interest in delivering cellular agents to infarcted myocardium to prevent postinfarction left ventricular remodeling. MRI can be effectively used to differentiate infarcted from healthy myocardium. MR-guided delivery of cellular agents/therapeutics is appealing because the therapeutics can be precisely targeted to the desired location within the infarct. In this study, a steerable intramyocardial injection catheter that can be actively tracked under MRI was developed and tested. The components of the catheter were arranged to form a loopless RF antenna receiver coil that enabled active tracking. Feasibility studies were performed in canine and porcine myocardial infarction models. Myocardial delayed-enhancement (MDE) imaging identified the infarcted myocardium, and real-time MRI was used to guide left ventricular catheterization from a carotid artery approach. The distal 35 cm of the catheter was seen under MRI with a bright signal at the distal tip of the catheter. The catheter was steered into position, the distal tip was apposed against the infarct, the needle was advanced, and a bolus of MR contrast agent and tissue marker dye was injected intramyocardially, as confirmed by imaging and postmortem histology. A pilot study involving intramyocardial delivery of magnetically labeled stem cells demonstrated the utility of the active injection catheter system.

111In oxine labelled mesenchymal stem cell SPECT after intravenous administration in myocardial infarction.

Mesenchymal stem cells (MSCs) have shown therapeutic potential if successfully delivered to the intended site of myocardial infarction. The purpose of this pilot study was to test the feasibility of 111In oxine labelling of MSCs and single photon emission computed tomography (SPECT) imaging after intravenous administration in a porcine model of myocardial infarction. Adult farm pigs (n=2) were subjected to closed chest experimental myocardial infarction. 111In oxine labelled MSCs (1 x 10(7) to 2 x 10(7) cells) were infused intravenously, and SPECT imaging was performed initially and on days 1, 2, 7 and 14. High quality SPECT images were obtained through 2 weeks of imaging. High initial MSC localization occurred in the lungs and slow progressive accumulation occurred in the liver, spleen and bone marrow. Renal activity was mild and persistent throughout imaging. No appreciable accumulation occurred in the myocardium. It is concluded that 111In oxine radiolabelling of MSCs is feasible, and in vivo imaging with SPECT provides a non-invasive method for sequentially monitoring cell trafficking with good spatial resolution. Because intravenous administration of MSCs results in significant lung activity that obscures the assessment of myocardial cell trafficking, alternative routes of administration should be investigated for this application.

Noninvasive quantification of total sodium concentrations in acute reperfused myocardial infarction using 23Na MRI.

The transport of sodium and potassium between the intra- and extracellular pools and the maintenance of the transmembrane concentration gradients are important to cell function and integrity. The early disruption of the sodium pump in myocardial infarction in response to the exhaustion of energy reserves following ischemia and reperfusion results in increased intracellular (and thus total) sodium levels. In this study a method for noninvasively quantifying myocardial sodium levels directly from sodium (23Na) MRI is presented. It was used to measure total myocardial sodium on a clinical 1.5T system in six normal dogs and five dogs with experimentally-induced myocardial infarction (MI). The technique was validated by comparing total sodium content measured by 23Na MRI with that measured by atomic absorption spectrophotometry (AAS) in biopsied tissue. Total sodium measured by 23Na MRI was significantly elevated in regions of infarction (81.3 +/- 14.3 mmol/kg wet wt, mean +/- SD) compared to noninfarcted myocardial tissue from both infarcted dogs (36.2 +/- 1.1, P < 0.001) and from normal controls (34.4 +/- 2.8, P < 0.0001). Myocardial tissue sodium content as measured by 23Na MRI did not vary regionally in the lateral, anterior, or inferior regions in normal hearts (ANOVA, P = NS). Sodium content measured by 23Na MRI agreed with the mean AAS estimates of 31.3 +/- 5.6 mmol/kg wet wt (P = NS) in normal hearts, and did not differ significantly from AAS measurements in MI (P = NS). Thus, local tissue sodium levels can be accurately quantified noninvasively using 23Na MRI in normal and acutely reperfused MI. The detection of regional myocardial sodium elevations may help differentiate viable from nonviable, infarcted tissue.

Superparamagnetic iron oxide MION as a contrast agent for sodium MRI in myocardial infarction.

An intravascular iron-based contrast agent was used as a sodium (23Na) MRI T2 relaxant in an effort to suppress the blood signal from the ventricular cavities in normal and infarcted canine myocardium in vivo. 23Na MRI signal decreases in blood were attributed to decreases in the fast (T2f) and slow (T2s) transverse relaxation components, which were quantified as a function of dose and MRI echo time (TE). In vivo 23Na MRI signal decreases up to 65% were noted in ventricular blood when imaging under dose and TE conditions of 10 mg/kg body weight and 5 ms, respectively. Contrast injection followed by subsequent 23Na MRI in canine myocardial infarction led to a clear delineation of the location of the injured tissue, as identified by postmortem triphenyltetrazolium chloride staining, and to an improvement in the contrast-to-noise ratio between the blood in the ventricular chamber and the infarcted tissue that was as high as 3.3-fold in the postcontrast images in comparison to the precontrast images.

A minimally invasive method for creating coronary stenosis in a swine model for MRI and SPECT imaging.

RATIONALE AND OBJECTIVES: To develop a less-invasive method for creating coronary stenosis in an animal model for the study of myocardial perfusion defects by using magnetic resonance imaging (MRI) and single-photon-emission computed tomography (SPECT). METHODS: Eleven farm pigs were instrumented with an MR-compatible coronary flow-reduction fitting in the left anterior descending coronary artery (LAD). These fittings were turned from a nylon rod, tapered from a maximum outer diameter of 3 mm, and drilled to a specified inner diameter (depending on the degree of coronary stenosis desired). The flow-reducing fittings were delivered over a coronary guidewire and advanced to a wedge position in the proximal LAD with an angioplasty catheter via a carotid artery approach. Perfusion determined by contrast-enhanced MRI at peak dipyridamole stress was compared with that obtained by 99mTc sestamibi SPECT. Radiolabeled microspheres were injected at rest, after stenosis implantation, and at peak pharmacological stress to establish the severity of the coronary lesion. RESULTS: Coronary stenosis was successfully created in seven animals. Mild coronary stenoses (<60%) were created in four animals. Significant coronary stenoses (80%-90%) were created in three animals. Thrombosis of the coronary flow-reducing fittings was observed in four animals, leading to sudden death in three animals and myocardial infarction in one animal. CONCLUSIONS: This method of angioplasty-guided, LAD coronary stenosis creation in a swine model presents a less-invasive alternative to open-chest techniques such as hydraulic occluders and ameroid constrictors.

Noninvasive assessment of myocardial stunning from short-term coronary occlusion using tagged magnetic resonance imaging.

Brief myocardial ischemia of less than 20 min duration, followed by reperfusion, is known to cause transient contractile dysfunction, often termed myocardial stunning. Tagged magnetic resonance imaging offers a noninvasive method that can be used to quantify this regional mechanical dysfunction in stunned myocardium. To this end, a closed-chest canine model of myocardial stunning was created by short-term (approximately 20-min) coronary occlusion, via inflation of an angioplasty balloon placed fluoroscopically in the left anterior descending (LAD) coronary, followed by reperfusion. Changes in myocardial strain before occlusion, during occlusion, and at 15 and 30 min after reperfusion were determined using repeated-measures analysis of variance. After instrumentation but before coronary occlusion, global reductions in myocardial strain were observed relative to animals that did not undergo coronary catheterization procedures. Declines of 46% and 49% in regional myocardial blood flow in the LAD and left circumflex bed, respectively, from preinstrumentation levels occurred due to coronary angiography and placement of a deflated angioplasty balloon in the LAD for 1 hr. During LAD occlusion, maximum myocardial shortening was significantly reduced in the anterior and anteroseptal regions of the left ventricular apex (i.e., ischemic region) but returned to baseline values by 30 min after reperfusion. No augmentation of myocardial function was observed in the nonischemic regions during occlusion or reperfusion. Thus, this noninvasive technique to evaluate myocardial ischemia demonstrated a graded response in myocardial function to ischemia and persistent regional dysfunction or "myocardial stunning" after short-term coronary occlusion.

Effect of dobutamine on regional left ventricular function measured by tagged magnetic resonance imaging in normal subjects.

The effect of inotropic stimulation on the pattern and magnitude of regional left ventricular contraction was studied using tagged magnetic resonance imaging to assess whether dobutamine exacerbates variation in regional contraction at rest. Dobutamine stress testing defines a normal response as a homogeneous increase in regional wall motion. In 8 normal subjects, 4 equally spaced left ventricular short-axis levels were imaged through systole using tagged magnetic resonance imaging. The baseline imaging sequence was repeated with 5-, 10-, 15-, and 20-microg/kg/min dobutamine infusion. Regional myocardial displacement, radial thickening, and circumferential shortening were measured. The left ventricle was analyzed by level (base to apex) and wall (septum, inferior, lateral, anterior). Dobutamine did not alter baseline regional functional heterogeneity. Dobutamine infusion resulted in a uniform increase in displacement, radial thickening, and circumferential shortening from baseline to 10-microg/kg/min infusion without additional increases at higher doses.

Intravascular MR-monitored balloon angioplasty: an in vivo feasibility study.

PURPOSE: To develop a new method for monitoring balloon angioplasty by using an intravascular magnetic resonance (MR) imaging technique. MATERIALS AND METHODS: Nine New Zealand White rabbits were used: seven for technique refinement, including surgery, device insertion, stenosis creation, and MR protocol development; and two for the final MR imaging of the balloon angioplasty. The in vivo experimental method involved insertion of a catheter antenna and a balloon catheter, via femoral arteriotomies bilaterally, into the target site of the upper abdominal aorta, where a stenosis was artificially created by binding a plastic cable tie. Then, the entire process of the dilation of the stenosis with balloon inflation was monitored under MR fluoroscopy. RESULTS: Catheter insertions were successful, and a 5-mm-long stenosis of the aorta was produced in all nine rabbits. Eight complete balloon angioplasty procedures were satisfactorily monitored and recorded, showing clearly the stenosis of the aorta at the beginning of the procedure, the dilation of the stenosis during the balloon inflation, and the complete opening of the stenosis after balloon dilation. CONCLUSION: Preliminary results of in vivo balloon angioplasty monitored with intravascular MR imaging are presented. MR fluoroscopy, based on the intravascular MR imaging technique, may represent a potential alternative to x-ray fluoroscopy for guiding interventional treatment of cardiovascular diseases.

Catheter-tracking FOV MR fluoroscopy.

Recent improvements in intravascular magnetic resonance imaging techniques mandate an accurate method of monitoring the introduction of MR catheter probes into the vessel of interest. For this purpose, a novel imaging protocol and a display method have been designed. First, a roadmap 3D image data set with standard pulse sequences is obtained using an external imaging coil. Subsequently, using very narrow rectangular-FOV fast-spoiled gradient recalled (SPGR), a movie of the percutaneous placement procedure of an MR catheter probe is acquired at a rate of 7.3 frames/second. In this protocol, the probe is used to transmit RF pulses and receive MR signal. A computer program was written for image unwrapping and for displaying the unwrapped movie frames on the roadmap image. In an alternative protocol, the movie frames in two projection angles were acquired in an interleaved fashion. Frames were unwrapped and combined with a 3D roadmap and displayed on a Silicon Graphics workstation equipped with stereovision goggles. Using these methods, percutaneous catheter placement in a phantom and a dog was examined. In conclusion, a new visualization technique for MR catheter placement is proposed. Combining this technique with high resolution intravascular MRI techniques may result in a very useful diagnostic tool for the evaluation of atherosclerosis and other vessel diseases.

Integrated MRI assessment of regional function and perfusion in canine myocardial infarction.

A single integrated examination using regional measurements of perfusion from contrast-enhanced MRI and three-dimensional (3D) strain from tissue-tagged MRI was developed to differentiate infarcted myocardium from adjacent tissue with functional abnormalities. Ten dogs were studied at baseline and 10 days after a 2-hour occlusion of the left anterior descending coronary artery (LAD). Strain was determined using a 3D finite element model. Two-dimensional measurements of hypoenhancing regions were highly correlated with myocardial viability (r = 0.96). Signal intensity versus time curves obtained from contrast-enhanced MRI were used for quantitative perfusion analysis. The remote and adjacent noninfarcted tissue of the dogs with LAD occlusion, as well as the infarcted tissue, exhibited abnormal deformation patterns as compared to normal dogs (positive predictive value (PPV) of strain determination of infarction = 66%). Integration of contrast-enhanced MRI results with 3D strain analysis enabled the delineation of the myocardial infarction (PPV = 100%) from functionally compromised myocardium. This integrated cardiac examination shows promise for noninvasive serial assessment of potentially jeopardized noninfarcted myocardium to study the process of infarct remodeling and expansion.

Right ventricular regional function using MR tagging: normals versus chronic pulmonary hypertension.

Right ventricular (RV) regional function, in both normal and diseased states, is not well characterized. Using 1D MR myocardial tagging, RV and septal intramyocardial segmental shortening was noninvasively measured in ten healthy subjects and in seven patients with chronic pulmonary hypertension. The normal RV free wall regional shortening was not uniform. A pattern of increasing RV free wall short-axis shortening was found from the RV outflow tract to the RV apex, and a more complex pattern of RV free wall long-axis shortening was observed. Both regional short- and long-axis shortening were globally reduced in pulmonary hypertension patients, with the greatest decreases in the RV outflow tract and in the basal septal wall region. Regional RV function can be quantitatively evaluated using MR tagging to determine the impact of chronic pulmonary hypertension on RV performance.

Myocardial perfusion and function in dogs with moderate coronary stenosis.

MRI studies of first-pass contrast enhancement with polylysine-Gd-DTPA and myocardial tagging using spatial modulation of magnetization (SPAMM) were performed to assess the feasibility of a combined regional myocardial blood flow and 2D deformation exam. Instrumented closed-chest dogs were imaged at a baseline control state (Cntl) followed by two interventions: moderate coronary stenosis (St) achieved by partial occlusion of the left anterior descending (LAD) and moderate coronary stenosis with dobutamine loading (StD). Hypoperfusion of the anterior region (ANT) of the myocardium (LAD distribution) relative to the posterior wall (POS) based on the upslope of the signal intensity time curve from the contrast-enhanced MR images was demonstrated only with dobutamine loading (ANT:POS Cntl = 1.077 +/- 0.15 versus ANT:POS StD = 0.477 +/- 0.11, P < 0.03) and was confirmed with radiolabeled microspheres measurements (ANT:POS Cntl = 1.18 +/- 0.2 ml/min/g versus ANT:POS StD = 0.44 +/- 0.1 ml/min/g; P < 0.002). Significant changes in regional myocardial shortening were only seen in the StD state (P < 0.02); the anterior region showed impaired myocardial shortening with dobutamine loading (P = NS), whereas the nonaffected POS region showed a marked increase in shortening when compared with Cntl (Cntl = 0.964 +/- 0.02 versus StD = 0.884 +/- 0.03; P < 0.001). These results demonstrate that an integrated quantitative assessment of regional myocardial function and semiquantitative assessment of myocardial blood flow can be performed noninvasively with ultrafast MRI.

Semi-automatic tracking of myocardial motion in MR tagged images.

Tissue tagging using magnetic resonance (MR) imaging has enabled quantitative noninvasive analysis of motion and deformation in vivo. One method for MR tissue tagging is Spatial Modulation of Magnetization (SPAMM). Manual detection and tracking of tissue tags by visual inspection remains a time-consuming and tedious process. The authors have developed an interactively guided semi-automated method of detecting and tracking tag intersections in cardiac MR images. A template matching approach combined with a novel adaptation of active contour modeling permits rapid analysis of MR images. The authors have validated their technique using MR SPAMM images of a silicone gel phantom with controlled deformations. Average discrepancy between theoretically predicted and semi-automatically selected tag intersections was 0.30 mm+/-0.17 [mean+/-SD, NS (P<0.05)]. Cardiac SPAMM images of normal volunteers and diseased patients also have been evaluated using the authors' technique.

Tracking and finite element analysis of stripe deformation in magnetic resonance tagging.

Magnetic resonance tissue tagging allows noninvasive in vivo measurement of soft tissue deformation. Planes of magnetic saturation are created, orthogonal to the imaging plane, which form dark lines (stripes) in the image. The authors describe a method for tracking stripe motion in the image plane, and show how this information can be incorporated into a finite element model of the underlying deformation. Human heart data were acquired from several imaging planes in different orientations and were combined using a deformable model of the left ventricle wall. Each tracked stripe point provided information on displacement orthogonal to the original tagging plane, i.e., a one-dimensional (1-D) constraint on the motion. Three-dimensional (3-D) motion and deformation was then reconstructed by fitting the model to the data constraints by linear least squares. The average root mean squared (rms) error between tracked stripe points and predicted model locations was 0.47 mm (n=3,100 points). In order to validate this method and quantify the errors involved, the authors applied it to images of a silicone gel phantom subjected to a known, well-controlled, 3-D deformation. The finite element strains obtained were compared to an analytic model of the deformation known to be accurate in the central axial plane of the phantom. The average rms errors were 6% in both the reconstructed shear strains and 16% in the reconstructed radial normal strain.