Vikram D. Kodibagkar

MR imaging of diffusion of 3He gas in healthy and diseased lungs

Hyperpolarized 3He gas MRI was used to form maps of the effective diffusivity of gas in human lungs. Images of diffusion as well as spin density are presented from a study of 11 healthy volunteers and 5 patients with severe emphysema. The effective rate of diffusion, De, of the gas is reduced by the alveolar walls; tissue destruction in emphysema is hypothesized to result in larger De. Indeed, the mean value of De in the emphysematous lungs is found here to be about 2.5 times that of healthy lungs, although still smaller than the unrestricted diffusivity of 3He in free air. Histograms of De values across coronal slices are presented. The results are discussed in terms of spatial variations, variations among individuals, healthy and diseased, and variations due to changes in lung volume. Magn Reson Med 44:174–179, 2000.

19F: A Versatile Reporter for Non-Invasive Physiology and Pharmacology Using Magnetic Resonance

The fluorine atom provides an exciting tool for diverse spectroscopic and imaging applications using Magnetic Resonance. The organic chemistry of fluorine is widely established and it can provide a stable moiety for interrogating many aspects of physiology and pharmacology in vivo. Strong NMR signal, minimal background signal and exquisite sensitivity to changes in the microenvironment have been exploited to design and apply diverse reporter molecules. Classes of agents are presented to investigate gene activity, pH, metal ion concentrations (e.g., Ca(2+), Mg(2+), Na(+)), oxygen tension, hypoxia, vascular flow and vascular volume. In addition to interrogating speciality reporter molecules, (19)F NMR may be used to trace the fate of fluorinated drugs, such as chemotherapeutics (e.g., 5-fluorouracil, gemcitabine), anesthetics (e.g., isoflurane, methoxyflurane) and neuroleptics. NMR can provide useful information through multiple parameters, including chemical shift, scalar coupling, chemical exchange and relaxation processes (R1 and R2). Indeed, the large chemical shift range (approximately 300 ppm) can allow multiple agents to be examined, simultaneously, using NMR spectroscopy or chemical shift selective imaging.

Vascular imaging of solid tumors in rats with a radioactive arsenic-labeled antibody that binds exposed phosphatidylserine.

Purpose: We recently reported that anionic phospholipids, principally phosphatidylserine, become exposed on the external surface of vascular endothelial cells in tumors, probably in response to oxidative stresses present in the tumor microenvironment. In the present study, we tested the hypothesis that a chimeric monoclonal antibody that binds phosphatidylserine could be labeled with radioactive arsenic isotopes and used for molecular imaging of solid tumors in rats. Experimental Design: Bavituximab was labeled with 74As (β+, T1/2 17.8 days) or 77As (β−, T1/2 1.6 days) using a novel procedure. The radionuclides of arsenic were selected because their long half-lives are consistent with the long biological half lives of antibodies in vivo and because their chemistry permits stable attachment to antibodies. The radiolabeled antibodies were tested for the ability to image subcutaneous Dunning prostate R3227-AT1 tumors in rats. Results: Clear images of the tumors were obtained using planar γ-scintigraphy and positron emission tomography. Biodistribution studies confirmed the specific localization of bavituximab to the tumors. The tumor-to-liver ratio 72 h after injection was 22 for bavituximab compared with 1.5 for an isotype-matched control chimeric antibody of irrelevant specificity. Immunohistochemical studies showed that the bavituximab was labeling the tumor vascular endothelium. Conclusions: These results show that radioarsenic-labeled bavituximab has potential as a new tool for imaging the vasculature of solid tumors.

Tumour oxygen dynamics measured simultaneously by near-infrared spectroscopy and 19F magnetic resonance imaging in rats*

Simultaneous near-infrared spectroscopy (NIRS) and magnetic resonance imaging (MRI) were used to investigate the correlation between tumour vascular oxygenation and tissue oxygen tension dynamics in rat breast 13762NF tumours with respect to hyperoxic gas breathing. NIRS directly detected global variations in the oxygenated haemoglobin concentration (Delta[HbO(2)]) within tumours and oxygen tension (pO(2)) maps were achieved using (19)F MRI of the reporter molecule hexafluorobenzene. Multiple correlations were examined between rates and magnitudes of vascular (Delta[HbO(2)]) and tissue (pO(2)) responses. Significant correlations were found between response to oxygen and carbogen breathing using either modality. Comparison of results for the two methods showed a correlation between the vascular perfusion rate ratio and the mean pO(2) values (R(2) > 0.7). The initial rates of increase of Delta[HbO(2)] and the slope of dynamic pO(2) response, d(pO(2))/dt, of well-oxygenated voxels in response to hyperoxic challenge were also correlated. These results demonstrate the feasibility of simultaneous measurements using NIRS and MRI. As expected, the rate of pO(2) response to oxygen is primarily dependent upon the well perfused rather than poorly perfused vasculature.

On the potential for molecular imaging with Cerenkov luminescence

Recent observation of optical luminescence due to beta decay from suitable radiotracers has led to the possible development of new preclinical optical imaging methods. The generation of photons that can be detected using instrumentation optimized for bioluminescence imaging has been putatively associated with the Cerenkov effect. We describe the simultaneous utilization of fluorescence reporters to convert Cerenkov luminescence to longer wavelengths for better tissue penetration and also for modulating the luminescence spectrum for potential molecular imaging strategies.

19F-NMR detection of lacZ gene expression via the enzymic hydrolysis of 2-fluoro-4-nitrophenyl β-D-galactopyranoside in vivo in PC3 prostate tumor xenografts in the mouse

Gene therapy shows promise for treating prostate cancer and has been evaluated in several clinical trials. A major challenge that remains is to establish a method for verifying transgene activity in situ. The lacZ gene encoding β‐galactosidase historically has been the most popular reporter gene for molecular biology. We have designed a 19F NMR approach to reveal lacZ gene expression by assessing β‐galactosidase (β‐gal) activity in vivo. The substrate 2‐fluoro‐4‐nitro‐phenyl β‐D‐galactopyranoside (OFPNPG) is readily hy‐drolyzed by β‐gal with a corresponding decrease in the 19F‐NMR signal from OFPNPG and the appearance of a new signal shifted 4–6 ppm upfield from the aglycone 2‐fluoro‐4‐nitrophenol (OFPNP). We report proof of principle in cultures of PC3 prostate cancer cells using 19F NMR spectroscopy and 19F chemical shift imaging. More importantly, we demonstrate for the first time the ability to differentiate wild‐type and lacZ‐expressing prostate tumor xenografts in mice using this approach.–Liu, L., Kodibagkar, V. D., Yu, J.‐X., Mason, R. P. 19F‐NMR detection of lacZ gene expression via the enzymic hydrolysis of 2‐fluoro‐4‐nitrophenyl β‐D‐galac‐topyranoside in vivo in PC3 prostate tumor. FASEB J. 21, 2014–2019 (2007)

Novel 1H NMR approach to quantitative tissue oximetry using hexamethyldisiloxane

19F NMR spin‐lattice relaxometry of hexafluorobenzene (HFB) has been shown to be a highly sensitive indicator of tumor oxygenation. In this study hexamethyldisiloxane (HMDSO) was identified as a proton NMR analog, and its potential as a probe for investigating dynamic changes in tissue oxygen tension (pO2) was evaluated. HMDSO has a single proton resonance (δ= –0.3 ppm) and the spin‐lattice relaxation rate, Rl (= 1/T1) exhibits a linear dependence on pO2: R1 (s–1) = 0.1126 + 0.0013* pO2 (torr) at 37°C. To demonstrate application in vivo, HMDSO was administered into healthy rat thigh muscle (100 μl) and tumors (50 μl). Local pO2 was determined by using pulse‐burst saturation recovery (PBSR) 1H NMR spectroscopy to assess R1. Water and fat signals were effectively suppressed by frequency‐selective excitation of the HMDSO resonance. Rat thigh muscle had a mean baseline pO2 of 35 ± 11 torr, with a typical stability of ±3 torr over 20 min, when the rats breathed air. Altering the inhaled gas to oxygen produced a significant increase in pO2 to 100–200 torr. In tumors, altering the inspired gas also produced significant (albeit generally smaller) changes. This new pO2 reporter molecule offers a potentially valuable new tool for investigating pO2 in vivo. Magn Reson Med, 2006.

Uncoupling hypoxia signaling from oxygen sensing in the liver results in hypoketotic hypoglycemic death

As the ultimate electron acceptor in oxidative phosphorylation, oxygen plays a critical role in metabolism. When oxygen levels drop, heterodimeric hypoxia-inducible factor (Hif) transcription factors become active and facilitate adaptation to hypoxia. Hif regulation by oxygen requires the protein von Hippel-Lindau (pVhl) and pVhl disruption results in constitutive Hif activation. The liver is a critical organ for metabolic homeostasis, and Vhl inactivation in hepatocytes results in a Hif-dependent shortening in life span. While albumin-Cre;VhlF/F mice develop hepatic steatosis and impaired fatty acid oxidation, the variable penetrance and unpredictable life expectancy has made the cause of death elusive. Using a system in which Vhl is acutely disrupted and a combination of ex vivo liver perfusion studies and in vivo oxygen measurements, we demonstrate that Vhl is essential for mitochondrial respiration in vivo. Adenovirus-Cre mediated acute Vhl disruption in the liver caused death within days. Deprived of pVhl, livers accumulated tryglicerides and circulating ketone and glucose levels dropped. The phenotype was reminiscent of inborn defects in fatty acid oxidation and of fasted PPARα-deficient mice and while death was unaffected by pharmacologic PPARα activation, it was delayed by glucose administration. Ex vivo liver perfusion analyses and acylcarnitine profiles showed mitochondrial impairment and a profound inhibition of liver ketone and glucose production. By contrast, other mitochondrial functions, such as ureagenesis, were unaffected. Oxygen consumption studies revealed a marked suppression of mitochondrial respiration, which, as determined by magnetic resonance oximetry in live mice, was accompanied by a corresponding increase in liver pO2. Importantly, simultaneous inactivation of Hif-1β suppressed liver steatosis and rescued the mice from death. These data demonstrate that constitutive Hif activation in mice is sufficient to suppress mitochondrial respiration in vivo and that no other pathway exists in the liver that can allow oxygen utilization when Hif is active precluding thereby metabolic collapse.

Quantitative tissue oxygen measurement in multiple organs using 19F MRI in a rat model

Measurement of individual organ tissue oxygen levels can provide information to help evaluate and optimize medical interventions in many areas including wound healing, resuscitation strategies, and cancer therapeutics. Echo planar 19F MRI has previously focused on tumor oxygen measurement at low oxygen levels (pO2) <30 mmHg. It uses the linear relationship between spin‐lattice relaxation rate (R1) of hexafluorobenzene (HFB) and pO2. The feasibility of this technique for a wider range of pO2values and individual organ tissue pO2 measurement was investigated in a rat model. Spin‐lattice relaxation times (T1= 1/R1) of hexafluorobenzene were measured using 19F saturation recovery echo planar imaging. Initial in vitro studies validated the linear relationship between R1 and pO2 from 0 to 760 mmHg oxygen partial pressure at 25, 37, and 41°C at 7 Tesla for hexafluorobenzene. In vivo experiments measured rat tissue oxygen (ptO2) levels of brain, kidney, liver, gut, muscle, and skin during inhalation of both 30 and 100% oxygen. All organ ptO2 values significantly increased with hyperoxia (P < 0.001). This study demonstrates that 19F MRI of hexafluorobenzene offers a feasible tool to measure regional ptO2 in vivo, and that hyperoxia significantly increases ptO2 of multiple organs in a rat model. Magn Reson Med, 2011.

Physical principles of quantitative nuclear magnetic resonance oximetry.

Over the years many techniques have been devised for the measurement of tissue oxygenation (oximetry). Oximetry using polarographic needle electrodes has long been considered a gold standard. Nuclear Magnetic Resonance (NMR) based oximetry uses exogenously administered reporter molecules such as perfluorocarbons to quantitatively interrogate oxygen tension (pO2). This technique has been successfully used in vivo in the preclinical setting and shows promise for clinical applications. NMR pO2 reporter molecules display a linear dependence of the spin lattice relaxation rate on pO2, which forms the basis of this technique. Physical principles of spin lattice relaxation of pO2 reporter molecules and the pO2 dependence of relaxation rate are discussed in this review. Practical considerations for choice of reporter molecules for in vivo measurements, general methodology and new developments are also described.