Depolymerizing self-immolative polymeric lanthanide chelates for vascular imaging.

Medical imaging is widely used clinically and in research to understand disease progression and monitor responses to therapies. Vascular imaging enables the study of vascular disease and therapy, but exogenous contrast agents are generally needed to distinguish the vasculature from surrounding soft tissues. Lanthanide-based agents are commonly employed in MRI, but are also of growing interest for micro-CT, as the position of their k-edges allows them to provide enhanced contrast and also to be employed in dual-energy micro-CT, a technique that can distinguish contrast-enhanced blood vessels from tissues such as bone. Small molecule Gd3+ chelates are available, but are excreted too rapidly. At the same time, a lack of rapid clearance from the body for long-circulating agents presents toxicity concerns. To address these challenges, we describe here the use of self-immolative polymers for the development of new degradable chelates that depolymerize completely from end-to-end following the cleavage of a single end-cap from the polymer terminus. We demonstrate that tuning the end-cap allows the rate of depolymerization to be controlled, while tuning the polymer length enables the polymer to exhibit long circulation times in the blood of mice. After successfully providing one hour of blood contrast, depolymerization led to excretion of the resulting small molecule chelates into the bladder. Despite the high doses required for micro-CT, the agents were well tolerated in mice. Thus, these self-immolative polymeric chelates provide a new platform for the development of medical imaging contrast agents. STATEMENT OF SIGNIFICANCE: Vascular imaging is used clinically to diagnose and monitor vascular disease and in research to understand the progression of disease and study responses to new therapies. For techniques such as magnetic resonance imaging and x-ray computed tomography (CT), long circulating contrast agents are needed to differentiate the vasculature from surrounding tissues. However, if these agents are not rapidly excreted from the body, they can lead to toxicity. We present here a new polymeric system that can chelate hundreds of lanthanide ions for imaging contrast and can circulate for one hour in the blood, but then after end-cap cleavage breaks down completely into small molecules for excretion. The successful application of this system in micro-CT in mice is demonstrated.

Periostin and CCN2 Scaffolds Promote the Wound Healing Response in the Skin of Diabetic Mice.

IMPACT STATEMENT: Nonhealing skin wounds remain a significant burden on health care systems, with diabetic patients 20 times as likely to undergo a lower extremity amputation due to impaired healing. Novel treatments that suppress the proinflammatory signature and induce the proliferative and remodeling phases are needed clinically. We demonstrate that the addition of periostin and CCN2 in a scaffold form increases closure rates of full-thickness skin wounds in diabetic mice, concomitant with enhanced angiogenesis. Our results demonstrate the efficacy of periostin- and CCN2-containing biomaterials to stimulate wound closure, which could represent a novel method for the treatment of diabetic skin wounds.

Dual-energy computed tomography using a gantry-based preclinical cone-beam microcomputed tomography scanner.

Dual-energy microcomputed tomography (DECT) can provide quantitative information about specific materials of interest, facilitating automated segmentation, and visualization of complex three-dimensional tissues. It is possible to implement DECT on currently available preclinical gantry-based cone-beam micro-CT scanners; however, optimal decomposition image quality requires customized spectral shaping (through added filtration), optimized acquisition protocols, and elimination of misregistration artifacts. We present a method for the fabrication of customized x-ray filters-in both shape and elemental composition-needed for spectral shaping. Fiducial markers, integrated within the sample holder, were used to ensure accurate co-registration between sequential low- and high-energy image volumes. The entire acquisition process was automated through the use of a motorized filter-exchange mechanism. We describe the design, implementation, and evaluation of a DECT system on a gantry-based-preclinical cone-beam micro-CT scanner.

Polymer Assembly Encapsulation of Lanthanide Nanoparticles as Contrast Agents for In Vivo Micro-CT.

Despite recent technological advancements in microcomputed tomography (micro-CT) and contrast agent development, preclinical contrast agents are still predominantly iodine-based. Higher contrast can be achieved when using elements with higher atomic numbers, such as lanthanides; lanthanides also have X-ray attenuation properties that are ideal for spectral CT. However, the formulation of lanthanide-based contrast agents at the high concentrations required for vascular imaging presents a significant challenge. In this work, we developed an erbium-based contrast agent that meets micro-CT imaging requirements, which include colloidal stability upon redispersion at high concentrations, evasion of rapid renal clearance, and circulation times of tens of minutes in small animals. Through systematic studies with poly(ethylene glycol) (PEG)-poly(propylene glycol), PEG-polycaprolactone, and PEG-poly(l-lactide) (PLA) block copolymers, the amphiphilic block copolymer PEG114-PLA53 was identified to be ideal for encapsulating oleate-coated lanthanide-based nanoparticles for in vivo intravenous administration. We were able to synthesize a contrast agent containing 100 mg/mL of erbium that could be redispersed into colloidally stable particles in saline after lyophilization. Contrast enhancement of over 250 HU was achieved in the blood pool for up to an hour, thereby meeting the requirements of live animal micro-CT.

Application of 3-d echocardiography and gated micro-computed tomography to assess cardiomyopathy in a mouse model of duchenne muscular dystrophy.

The purpose of this study was to measure changes in cardiac function as cardiomyopathy progresses in a mouse model of Duchenne muscular dystrophy using 3-D ECG-gated echocardiography. This study is the first to correlate cardiac volumes acquired using 3-D echocardiography with those acquired using retrospectively gated micro-computed tomography (CT). Both were further compared with standard M-mode echocardiography and histologic analyses. We found that although each modality measures a decrease in cardiac function as disease progresses in mdx/utrn(-/-) mice (n = 5) compared with healthy C57BL/6 mice (n = 8), 3-D echocardiography has higher agreement with gold-standard measurements acquired by gated micro-CT, with little standard deviation between measurements. M-Mode echocardiography measurements, in comparison, exhibit considerably greater variability and user bias. Given the radiation dose associated with micro-CT and the geometric assumptions made in M-mode echocardiography to calculate ventricular volume, we suggest that use of 3-D echocardiography has important advantages that may allow for the measurement of early disease changes that occur before overt cardiomyopathy.

X-ray dose delivered during a longitudinal micro-CT study has no adverse effect on cardiac and pulmonary tissue in C57BL/6 mice.

BACKGROUND: Micro-computed tomography (micro-CT) offers numerous advantages for small animal imaging, including the ability to monitor the same animals throughout a longitudinal study. However, concerns are often raised regarding the effects of X-ray dose accumulated over the course of the experiment. PURPOSE: To scan C57BL/6 mice multiple times per week for 6 weeks, in order to determine the effect of the cumulative dose on pulmonary and cardiac tissue at the end of the study. MATERIAL AND METHODS: C57BL/6 male mice were split into two groups (irradiated group = 10, control group = 10). The irradiated group was scanned (80 kVp/50 mA) three times weekly for 6 weeks, resulting in a weekly dose of 0.84 Gy, and a total study dose of 5.04 Gy. The control group was scanned on the final week. Scans from week 6 were reconstructed and the lungs and heart were analyzed. RESULTS: Overall, there was no significant difference in lung volume or lung density or in left ventricular volume or ejection fraction between the control group and the irradiated group. Histological samples taken from excised lung and myocardial tissue also showed no evidence of inflammation or fibrosis in the irradiated group. CONCLUSION: This study demonstrated that a 5 Gy X-ray dose accumulated over 6 weeks during a longitudinal micro-CT study had no significant effects on the pulmonary and myocardial tissue of C57BL/6 mice. As a result, the many advantages of micro-CT imaging, including rapid acquisition of high-resolution, isotropic images in free-breathing mice, can be taken advantage of in longitudinal studies without concern for negative dose-related effects.

Rapid microcomputed tomography suggests cardiac enlargement occurs during conductance catheter measurements in mice.

Conductance catheters (CC) represent an established method of determining cardiac function in mice; however, the potentially detrimental effects a catheter may have on the mouse heart have never been evaluated. The present study takes advantage of rapid three-dimensional (3D) microcomputed tomography (CT) to compare simultaneously acquired micro-CT and CC measurements of left ventricular (LV) volumes in healthy and infarcted mice and to determine changes in LV volume and function associated with CC insertion. LV volumes were measured in C57BL/6 mice (10 healthy, 10 infarcted, 2% isoflurane anesthesia) using a 1.4-Fr Millar CC. 3D micro-CT images of each mouse were acquired before CC insertion as well as during catheterization. Each CT scan produced high-resolution images throughout the entire cardiac cycle in <1 min, enabling accurate volume measurements as well as direct visualization of the CC within the LV. Bland-Altman analysis demonstrated that CC measurements underestimate volume compared with CT measurements in both healthy [bias of -18.4 and -28.9 µl for end-systolic (ESV) and end-diastolic volume (EDV), respectively] and infarcted mice (ESV = -51.6 µl and EDV = -71.7 µl); underestimation was attributed to the off-center placement of the catheter. Individual evaluation of each heart revealed LV dilation following CC insertion in 40% of mice in each group. No change in ejection fraction was observed, suggesting the enlargement was caused by volume overload associated with disruption of the papillary muscles or chords. The enlargement witnessed was not significant; however, the results suggest the potential for CC insertion to detrimentally affect mouse myocardium, necessitating further investigation.

Evaluation of eXIA 160 cardiac-related enhancement in C57BL/6 and BALB/c mice using micro-CT.

Evaluation of cardiovascular function in mice using micro-CT requires that a contrast agent be administered to differentiate the blood from the myocardium. eXIA 160, an aqueous colloidal poly-disperse contrast agent with a high iodine concentration (160 mg I ml(-1)), creates strong contrast between blood and tissue with a low injection volume. In this study, the blood-pool enhancement time-course of eXIA 160 is monitored over a 48 h period to determine its optimal use during cardiac function studies in C57BL/6 and BALB/c mice. Eight-second scans were performed (80 kV(p), 110 mA) using the GE Locus Ultra micro-CT scanner. Six C57BL/6 and six BALB/c male mice (22-24 g) were injected via tail vein with 5 µl g(-1) body weight eXIA 160. A precontrast scan was performed; following injection, mice were scanned at 5, 15, 30, 45 and 60 min, and 2, 4, 8, 12, 24 and 48 h. Images were reconstructed, and enhancement-time curves were generated for each of the following tissues: left ventricle (LV), myocardium, liver, spleen, renal cortex, bladder and brown adipose tissue. The highest contrast in the LV occurred at 5 min in both strains (~670 HU above precontrast value). Uptake of the contrast agent by the myocardium was also observed: myocardial tissue showed increasing enhancement over a 4 h period in both strains, remaining even once the contrast was eliminated from the vasculature. In both C57BL/6 and BALB/c strains, eXIA 160 provided high contrast between blood and myocardial tissue for a period of 30 min following injection. Notably, this contrast agent was also taken up by the myocardium and provided continued enhancement when it was eliminated from the blood, making LV wall motion studies possible. In conclusion, eXIA 160, with its high iodine concentration and targeted tissue uptake characteristics, is an ideal agent to use when evaluating cardiovascular function in mice.

Functional neoangiogenesis imaging of genetically engineered mouse prostate cancer using three-dimensional power Doppler ultrasound.

We report the first application of high-frequency three-dimensional power Doppler ultrasound imaging in a genetically engineered mouse (GEM) prostate cancer model. We show that the technology sensitively and specifically depicts functional neoangiogenic blood flow because little or no flow is measurable in normal prostate tissue or tumors smaller than 2-3 mm diameter, the neoangiogenesis "switch-on" size. Vascular structures depicted by power Doppler were verified using Microfil-enhanced micro-computed tomography (micro-CT) and by correlation with microvessel distributions measured by immunohistochemistry and enhanced vascularity visualized by confocal microscopy in two GEM models [transgenic adenocarcinoma of the mouse prostate (TRAMP) and PSP94 gene-directed transgenic mouse adenocarcinoma of the prostate (PSP-TGMAP)]. Four distinct phases of neoangiogenesis in cancer development were observed, specifically, (a) an early latent phase; (b) establishment of a peripheral capsular vascular structure as a neoangiogenesis initiation site; (c) a peak in tumor vascularity that occurs before aggressive tumor growth; and (d) rapid tumor growth accompanied by decreasing vascularity. Microsurgical interventions mimicking local delivery of antiangiogenesis drugs were done by ligating arteries upstream from feeder vessels branching to the prostate. Microsurgery produced an immediate reduction of tumor blood flow, and flow remained low from 1 h to 2 weeks or longer after treatment. Power Doppler, in conjunction with micro-CT, showed that the tumors recruit secondary blood supplies from nearby vessels, which likely accounts for the continued growth of the tumors after surgery. The microsurgical model represents an advanced angiogenic prostate cancer stage in GEM mice corresponding to clinically defined hormone-refractory prostate cancer. Three-dimensional power Doppler imaging is completely noninvasive and will facilitate basic and preclinical research on neoangiogenesis in live animal models.

Evaluation of an algorithm for semiautomated segmentation of thin tissue layers in high-frequency ultrasound images.

An algorithm consisting of speckle reduction by median filtering, contrast enhancement using top- and bottom-hat morphological filters, and segmentation with a discrete dynamic contour (DDC) model was implemented for nondestructive measurements of soft tissue layer thickness. Algorithm performance was evaluated by segmenting simulated images of three-layer phantoms and high-frequency (40 MHz) ultrasound images of porcine aortic valve cusps in vitro. The simulations demonstrated the necessity of the median and morphological filtering steps and enabled testing of user-specified parameters of the morphological filters and DDC model. In the experiments, six cusps were imaged in coronary perfusion solution (CPS) then in distilled water to test the algorithm's sensitivity to changes in the dimensions of thin tissue layers. Significant increases in the thickness of the fibrosa, spongiosa, and ventricularis layers, by 53.5% (p < 0.001), 88.5% (p < 0.001), and 35.1% (p = 0.033), respectively, were observed when the specimens were submerged in water. The intraobserver coefficient of variation of repeated thickness estimates ranged from 0.044 for the fibrosa in water to 0.164 for the spongiosa in CPS. Segmentation accuracy and variability depended on the thickness and contrast of the layers, but the modest variability provides confidence in the thickness measurements.

Three-dimensional visualization and thickness estimation of aortic valve cusps using high-frequency ultrasound.

High-frequency ultrasound techniques are introduced for three-dimensional imaging and thickness estimation of fresh heart valve cusps. Images of porcine aortic valve specimens were acquired within a 12 x 8 x 8 mm3 volume using a VisualSonics VS40 micro-imaging system operating at a 40 MHz centre frequency. Two image volumes were obtained from each of six left coronary cusps. One volume was acquired with the specimen submerged in distilled water and the second volume was acquired through either Hanks physiologic solution or coronary perfusion solution (CPS). The fibrosa, spongiosa and ventricularis were most readily distinguished when the specimen was imaged in distilled water. Colour thickness maps were computed from B-mode image data, and the mean and standard deviations of the thickness were determined for each cusp. In 11 of 12 trials, the image analysis algorithm yielded valid thickness estimates over greater than 98% of the region examined. Mean thickness estimates obtained with specimens submerged in Hanks solution or CPS ranged from 0.66 to 1.03 mm, and submersion in distilled water increased the mean thickness by 20-40%. This observation suggests that the cusps osmotically absorbed water. Information provided by high-frequency ultrasound is expected be valuable for characterizing the morphological properties of heart valves.