In Vivo Mapping of Myocardial Injury Outside the Infarct Zone: Tissue at an Intermediate Pathological State.

BACKGROUND: The goal was to determine the feasibility of mapping the injured-but-not-infarcted myocardium using 99mTc-duramycin in the postischemic heart, with spatial information for its characterization as a pathophysiologically intermediate tissue, which is neither normal nor infarcted. METHODS AND RESULTS: Coronary occlusion was conducted in Sprague Dawley rats with preconditioning and 30-minute ligation. In vivo single-photon emission computed tomography was acquired after 3 hours (n=6) using 99mTc-duramycin, a phosphatidylethanolamine-specific radiopharmaceutical. The 99mTc-duramycin+ areas were compared with infarct and area-at-risk (n=8). Cardiomyocytes and endothelial cells were isolated for gene expression profiling. Cardiac function was measured with echocardiography (n=6) at 4 weeks. In vivo imaging with 99mTc-duramycin identified the infarct (3.9±2.4% of the left ventricle and an extensive area 23.7±2.2% of the left ventricle) with diffuse signal outside the infarct, which is pathologically between normal and infarcted (apoptosis 1.8±1.6, 8.9±4.2, 13.6±3.8%; VCAM-1 [vascular cell adhesion molecule 1] 3.2±0.8, 9.8±4.1, 15.9±4.2/mm2; tyrosine hydroxylase 14.9±2.8, 8.6±4.4, 5.6±2.2/mm2), with heterogeneous changes including scattered micronecrosis, wavy myofibrils, hydropic change, and glycogen accumulation. The 99mTc-duramycin+ tissue is quantitatively smaller than the area-at-risk (26.7% versus 34.4% of the left ventricle, P=0.008). Compared with infarct, gene expression in the 99mTc-duramycin+-noninfarct tissue indicated a greater prosurvival ratio (BCL2/BAX [B-cell lymphoma 2/BCL2-associated X] 7.8 versus 5.7 [cardiomyocytes], 3.7 versus 3.2 [endothelial]), and an upregulation of ion channels in electrophysiology. There was decreased contractility at 4 weeks (regional fractional shortening -8.6%, P<0.05; circumferential strain -52.9%, P<0.05). CONCLUSIONS: The injured-but-not-infarcted tissue, being an intermediate zone between normal and infarct, is mapped in vivo using phosphatidylethanolamine-based imaging. The intermediate zone contributes significantly to cardiac dysfunction.

Preclinical Applications of Multi-Platform Imaging in Animal Models of Cancer.

In animal models of cancer, oncologic imaging has evolved from a simple assessment of tumor location and size to sophisticated multimodality exploration of molecular, physiologic, genetic, immunologic, and biochemical events at microscopic to macroscopic levels, performed noninvasively and sometimes in real time. Here, we briefly review animal imaging technology and molecular imaging probes together with selected applications from recent literature. Fast and sensitive optical imaging is primarily used to track luciferase-expressing tumor cells, image molecular targets with fluorescence probes, and to report on metabolic and physiologic phenotypes using smart switchable luminescent probes. MicroPET/single-photon emission CT have proven to be two of the most translational modalities for molecular and metabolic imaging of cancers: immuno-PET is a promising and rapidly evolving area of imaging research. Sophisticated MRI techniques provide high-resolution images of small metastases, tumor inflammation, perfusion, oxygenation, and acidity. Disseminated tumors to the bone and lung are easily detected by microCT, while ultrasound provides real-time visualization of tumor vasculature and perfusion. Recently available photoacoustic imaging provides real-time evaluation of vascular patency, oxygenation, and nanoparticle distributions. New hybrid instruments, such as PET-MRI, promise more convenient combination of the capabilities of each modality, enabling enhanced research efficacy and throughput.

Whole-body Imaging of Cell Death Provides a Systemic, Minimally Invasive, Dynamic, and Near-real Time Indicator for Chemotherapeutic Drug Toxicity.

PURPOSE: Response to toxicity in chemotherapies varies considerably from tissue to tissue and from patient to patient. An ability to monitor the tissue damage done by chemotherapy may have a profound impact on treatment and prognosis allowing for a proactive management in understanding and mitigating such events. For the first time, we investigated the feasibility of using whole-body imaging to map chemotherapeutic drug-induced toxicity on an individual basis. EXPERIMENTAL DESIGN: In a preclinical proof-of-concept, rats were treated with a single clinical dose of cyclophosphamide, methotrexate, or cisplatin. In vivo whole-body imaging data were acquired using 99mTc-duramycin, which identifies dead and dying cells as an unambiguous marker for tissue injury in susceptible organs. Imaging results were cross-validated using quantitative ex vivo measurements and histopathology and compared with standard blood and serum panels for toxicology. RESULTS: The in vivo whole-body imaging data detected widespread changes, where spatially heterogeneous toxic effects were identified across different tissues, within substructures of organs, as well as among different individuals. The signal changes were consistent with established toxicity profiles of these chemotherapeutic drugs. Apart from generating a map of susceptible tissues, this in vivo imaging approach was more sensitive compared with conventional blood and serum markers used in toxicology. Also, repeated imaging during the acute period after drug treatment captured different kinetics of tissue injury among susceptible organs in males and females. CONCLUSIONS: This novel and highly translational imaging approach shows promise in optimizing therapeutic decisions by detecting and managing drug toxicity on a personalized basis.Toxicity to normal tissues is a significant limitation in chemotherapies. This work demonstrated an in vivo imaging-based approach for characterizing toxicity-induced tissue injury in a systemic, dynamic, and near-real time fashion. This novel approach shows promise in optimizing therapeutic decisions by monitoring drug toxicity on a personalized basis.

Flexible Transient Optical Waveguides and Surface-Wave Biosensors Constructed from Monocrystalline Silicon.

Optical technologies offer important capabilities in both biological research and clinical care. Recent interest is in implantable devices that provide intimate optical coupling to biological tissues for a finite time period and then undergo full bioresorption into benign products, thereby serving as temporary implants for diagnosis and/or therapy. The results presented here establish a silicon-based, bioresorbable photonic platform that relies on thin filaments of monocrystalline silicon encapsulated by polymers as flexible, transient optical waveguides for accurate light delivery and sensing at targeted sites in biological systems. Comprehensive studies of the mechanical and optical properties associated with bending and unfurling the waveguides from wafer-scale sources of materials establish general guidelines in fabrication and design. Monitoring biochemical species such as glucose and tracking physiological parameters such as oxygen saturation using near-infrared spectroscopic methods demonstrate modes of utility in biomedicine. These concepts provide versatile capabilities in biomedical diagnosis, therapy, deep-tissue imaging, and surgery, and suggest a broad range of opportunities for silicon photonics in bioresorbable technologies.

Gd(III)-labeled peptide nanofibers for reporting on biomaterial localization in vivo.

Bioactive supramolecular nanostructures are of great importance in regenerative medicine and the development of novel targeted therapies. In order to use supramolecular chemistry to design such nanostructures, it is extremely important to track their fate in vivo through the use of molecular imaging strategies. Peptide amphiphiles (PAs) are known to generate a wide array of supramolecular nanostructures, and there is extensive literature on their use in areas such as tissue regeneration and therapies for disease. We report here on a series of PA molecules based on the well-established ß-sheet amino acid sequence V3A3 conjugated to macrocyclic Gd(III) labels for magnetic resonance imaging (MRI). These conjugates were shown to form cylindrical supramolecular assemblies using cryogenic transmission electron microscopy and small-angle X-ray scattering. Using nuclear magnetic relaxation dispersion analysis, we observed that thermal annealing of the nanostructures led to a decrease in water exchange lifetime (τm) of hundreds of nanoseconds only for molecules that self-assemble into nanofibers of high aspect ratio. We interpret this decrease to indicate more solvent exposure to the paramagnetic moiety on annealing, resulting in faster water exchange within angstroms of the macrocycle. We hypothesize that faster water exchange in the nanofiber-forming PAs arises from the dehydration and increase in packing density on annealing. Two of the self-assembling conjugates were selected for imaging PAs after intramuscular injections of the PA C16V3A3E3-NH2 in the tibialis anterior muscle of a murine model. Needle tracts were clearly discernible with MRI at 4 days postinjection. This work establishes Gd(III) macrocycle-conjugated peptide amphiphiles as effective tracking agents for peptide amphiphile materials in vivo over the timescale of days.

PGC1α-mediated mitofusin-2 deficiency in female rats and humans with pulmonary arterial hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is a lethal, female-predominant, vascular disease. Pathologic changes in PA smooth muscle cells (PASMC) include excessive proliferation, apoptosis-resistance, and mitochondrial fragmentation. Activation of dynamin-related protein increases mitotic fission and promotes this proliferation-apoptosis imbalance. The contribution of decreased fusion and reduced mitofusin-2 (MFN2) expression to PAH is unknown. OBJECTIVES: We hypothesize that decreased MFN2 expression promotes mitochondrial fragmentation, increases proliferation, and impairs apoptosis. The role of MFN2's transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), was assessed. MFN2 therapy was tested in PAH PASMC and in models of PAH. METHODS: Fusion and fission mediators were measured in lungs and PASMC from patients with PAH and female rats with monocrotaline or chronic hypoxia+Sugen-5416 (CH+SU) PAH. The effects of adenoviral mitofusin-2 (Ad-MFN2) overexpression were measured in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS: In normal PASMC, siMFN2 reduced expression of MFN2 and PGC1α; conversely, siPGC1α reduced PGC1α and MFN2 expression. Both interventions caused mitochondrial fragmentation. siMFN2 increased proliferation. In rodent and human PAH PASMC, MFN2 and PGC1α were decreased and mitochondria were fragmented. Ad-MFN2 increased fusion, reduced proliferation, and increased apoptosis in human PAH and CH+SU. In CH+SU, Ad-MFN2 improved walking distance (381 ± 35 vs. 245 ± 39 m; P < 0.05); decreased pulmonary vascular resistance (0.18 ± 0.02 vs. 0.38 ± 0.14 mm Hg/ml/min; P < 0.05); and decreased PA medial thickness (14.5 ± 0.8 vs. 19 ± 1.7%; P < 0.05). Lung vascularity was increased by MFN2. CONCLUSIONS: Decreased expression of MFN2 and PGC1α contribute to mitochondrial fragmentation and a proliferation-apoptosis imbalance in human and experimental PAH. Augmenting MFN2 has therapeutic benefit in human and experimental PAH.

High-resolution MRI of excised human prostate specimens acquired with 9.4T in detection and identification of cancers: validation of a technique.

PURPOSE: To evaluate feasibility of high-resolution, high-field ex vivo prostate magnetic resonance imaging (MRI) as an aid to guide pathologists' examination and develop in vivo MRI methods. MATERIALS AND METHODS: Unfixed excised prostatectomy specimens (n = 9) were obtained and imaged immediately after radical prostatectomy under an Institutional Review Board-approved protocol. High-resolution T2-weighted (T2W) MRI of specimens were acquired with a Bruker 9.4 T scanner to correlate with whole-mount histology. Additionally, T2 and apparent diffusion coefficient (ADC) maps were generated. RESULTS: By visual inspection of the nine prostate specimens imaged, high-resolution T2W MRI showed improved anatomical detail compared to published low-resolution images acquired at 4 T as published by other investigators. Benign prostatic hyperplasia, adenocarcinomas, curvilinear duct architecture distortion due to adenocarcinomas, and normal radial duct distribution were readily identified. T2 was ≈10 msec longer (P < 0.03) and the ADC was ≈1.4 times larger (P < 0.002) in the normal peripheral zone compared to the peripheral zone with prostate cancer. CONCLUSION: Differences in T2 and ADC between benign and malignant tissue are consistent with in vivo data. High-resolution, high-field MRI has the potential to improve the detection and identification of prostate structures. The protocols and techniques developed in this study could augment routine pathological analysis of surgical specimens and guide treatment of prostate cancer patients.

HiSStology: high spectral and spatial resolution magnetic resonance imaging detection of vasculature validated by histology and micro-computed tomography.

High spectral and spatial resolution (HiSS) data, acquired with echo-planar spectroscopic imaging (EPSI), can be used to acquire water spectra from each small image voxel. These images are sensitive to changes in local susceptibility caused by superparamagnetic iron oxide particles (SPIO); therefore, we hypothesized that images derived from HiSS data are very sensitive to tumor neovasculature following injection of SPIO. Accurate image registration was used to validate HiSS detection of neovasculature with histology and micro-computed tomographic (microCT) angiography. Athymic nude mice and Copenhagen rats were inoculated with Dunning AT6.1 prostate tumor cells in the right hind limb. The tumor region was imaged pre- and post-intravenous injection of SPIO. Three-dimensional assemblies of the CD31-stained histologic slices of the mouse legs and the microCT images of the rat vascular casts were registered with EPSI. The average distance between HiSS-predicted regions of high vascular density on magnetic resonance imaging and CD31-stained regions on histology was 200 µm. Similarly, vessels identified by HiSS in the rat images coincided with vasculature in the registered microCT image. The data demonstrate a strong correlation between tumor vasculature identified using HiSS and two gold standards: histology and microCT angiography.

Where it's at really matters: in situ in vivo vascular endothelial growth factor spatially correlates with electron paramagnetic resonance pO2 images in tumors of living mice.

PURPOSE: Tumor microenvironments show remarkable tumor pO(2) heterogeneity, as seen in prior EPR pO(2) images (EPROI). pO(2) correlation with hypoxia response proteins is frustrated by large rapid pO(2) changes with position. PROCEDURES: To overcome this limitation, biopsies stereotactically located in the EPROI were used to explore the relationship between vascular endothelial growth factor A (VEGF) concentrations in living mouse tumors and the local EPROI pO(2). RESULTS: Quantitative ELISA VEGF concentrations correlated (p = 0.0068 to 0.019) with mean pO(2), median pO(2), and the fraction of voxels in the biopsy volume with pO(2) less than 3, 6, and 10 Torr. CONCLUSIONS: This validates EPROI hypoxic fractions at the molecular level and provides a new paradigm for the assessment of the relationship, in vivo, between hypoxia and hypoxia response proteins. When translated to human subjects, this will enhance understanding of human tumor pathophysiology and cancer response to therapy.

Electron paramagnetic resonance oxygen imaging of a rabbit tumor using localized spin probe delivery.

PURPOSE: Application of in vivo electron paramagnetic resonance (EPR) oxygen imaging (EPROI) to tumors larger than those of mice requires development of both instrumental and medical aspects of imaging. METHODS: 250 MHz EPR oxygen imaging was performed using a loop-gap resonator with a volume exceeding 100 cm3. The paramagnetic spin probe was injected directly into the femoral artery feeding the rabbit leg/tumor. RESULTS: The authors present continuous wave and electron spin echo EPR oxygen images of a large size (4 cm) VX-2 tumor located on the leg of a New Zealand white rabbit. CONCLUSIONS: This study demonstrates the feasibility of continuous wave and electron spin echo oxygen imaging modalities for investigation of volumes of tumor and normal tissue relevant to large animals. The injection of the spin probe directly into the artery feeding a rabbit leg will allow one to reduce, by over one order of magnitude, the amount of spin probe used as compared to whole animal i.v. injection.

Characterization of response to radiation mediated gene therapy by means of multimodality imaging.

Imaging techniques are under development to facilitate early analysis of spatial patterns of tumor response to combined radiation and antivascular gene therapy. A genetically modified, replication defective adenoviral vector (Ad.EGR-TNFalpha), injected intratumorally, mediates infected cells to express tumor necrosis factor alpha (TNFalpha), which is increased after exposure to radiation. The goal of this study was to characterize an image based "signature" for response to this combined radiation and gene therapy in mice with human prostate xenografts. This study is part of an imaged guided therapy project where such a signature would be useful in guiding subsequent treatments. Changes in the tumor micro-environment were assessed using MRI registered with electron paramagnetic resonance imaging which provides images of tissue oxygenation. Dynamic contrast-enhanced MRI was used to assess tissue perfusion. When compared with null vector (control) treatment, the ratio of contrast agent (Gd-DTPA-BMA) washout rate to uptake rate was lower (P = 0.001) after treatment, suggesting a more balanced perfusion. Concomitantly, oxygenation significantly increased in the treated animals and decreased or did not change in the control animals (P < 0.025). This is the first report of minimally invasive, quantitative, absolute oxygen measurements correlated with tissue perfusion in vivo.

Electron paramagnetic resonance oxygen image hypoxic fraction plus radiation dose strongly correlates with tumor cure in FSa fibrosarcomas.

PURPOSE: Tumor hypoxia has long been known to produce resistance to radiation. In this study, electron paramagnetic resonance (EPR) oxygen imaging was investigated for its power to predict the success of tumor control according to tumor oxygenation level and radiation dose. METHODS AND MATERIALS: A total of 34 EPR oxygen images were obtained from the legs of C3H mice bearing 0.5-cm(3) FSa fibrosarcomas under both normal (air breathing) and clamped tumor conditions. Under the same conditions as those during which the images were obtained, the tumors were irradiated to a variety of doses near the FSa dose at which 50% of tumors were cured. Tumor tissue was distinguished from normal tissue using co-registration of the EPR oxygen images with spin-echo magnetic resonance imaging of the tumor and/or stereotactic localization. The tumor voxel statistics in the EPR oxygen image included the mean and median partial pressure of oxygen and the fraction of tumor voxels below the specified partial pressure of oxygen values of 3, 6, and 10 mm Hg. Bivariate logistic regression analysis using the radiation dose and each of the EPR oxygen image statistics to determine which best separated treatment failure from success. RESULTS: The measurements of the dose at which 50% of tumors were cured were similar to those found in published data for this syngeneic tumor. Bivariate analysis of 34 tumors demonstrated that tumor cure correlated with dose (p = 0.004) and with a <10 mm Hg hypoxic fraction (p = 0.023). CONCLUSION: Our results have shown that, together, radiation dose and EPR image hypoxic fraction separate the population of FSa fibrosarcomas that are cured from those that fail, thus predicting curability.

Immobilization Using Dental Material Casts Facilitates Accurate Serial and Multimodality Small Animal Imaging.

Custom disposable patient immobilization systems that conform to the patient's body contours are commonly used to facilitate accurate repeated patient setup for imaging and treatment in radiation therapy. However, in small-animal imaging, immobilization is often overlooked or done in a way that is not conducive to reproducible positioning. This has a negative impact on the potential for accurate analysis of serial or multimodality imaging. We present the use of vinyl polysiloxane dental impression material for immobilization of mice for imaging. Four different materials were examined to identify any potential artifacts using magnetic resonance techniques. A water phantom placed inside the cast was used at 4.7 T with magnetic resonance imaging and showed no effect at the center of the image when compared with images without the cast. A negligible effect was seen near the ends of the coil. Each material had no detectable signal using electron paramagnetic resonance imaging at 9 mT. The use of dental material also greatly enhances the use of fiducial markers that can be embedded in the mold. Therefore, image registration is simplified as the immobilization of the animal and fiducials together helps in translating from one image coordinate system to another.

Reduction of image artifacts in mice by bladder flushing with a novel double-lumen urethral catheter.

In electron paramagnetic resonance imaging (EPRI), the accumulation of contrast agent in the bladder can create a very large source of signal, often far greater than that of the organ of interest. Mouse model images have become increasingly important in preclinical testing. To minimize bladder accumulation on mouse images, we developed a novel, minimally invasive, MRI/EPRI-friendly procedure for flushing a female mouse bladder. It is also applicable to other imaging techniques, for example, PET, SPECT, etc., where contrast agent accumulation in the bladder is also undesirable. A double-lumen urethral catheter was developed, using a standard IV catheter with a silicone tube extension, having a polyethylene tube threaded into the IV catheter. Flushing of the bladder provides a substantial reduction in artifacts, as shown in images of tumors in mice.