Rami R. Hallac

New frontiers and developing applications in 19F NMR

1.1 Overview 19F NMR is gaining interest as a tool for diverse physiological and pharmaceutical investigations as evidenced by new reporter molecules and detection strategies. The relatively high sensitivity of 19F and lack of interfering background signal in the body have enabled the observation of exogenously administered agents and their metabolites. The 19F nucleus exhibits a large chemical shift range (~300 ppm), which is exquisitely sensitive to the electronic microenvironment, and many reporter molecules have been developed. In addition to chemical shift studies, relaxation processes (R1 and R2), and chemical exchange have also been tailored to be responsive to a parameter of interest such as pO2, pH, metal ion concentrations, transgene/enzyme activity or hypoxia. Recent emphasis has been on enhancing signal sensitivity and developing novel response mechanisms to sense environmental parameters. This review focuses on quantitative tissue oximetry, detection of ions including pH, and detection of enzyme activity. Fluorine NMR has been widely exploited for laboratory investigations and is garnering developing use in pre-clinical applications for small animals in vivo.

Correlations of noninvasive BOLD and TOLD MRI with pO2 and relevance to tumor radiation response

To examine the potential use of blood oxygenation level dependent (BOLD) and tissue oxygenation level dependent (TOLD) contrast MRI to assess tumor oxygenation and predict radiation response.

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.

Oxygenation in cervical cancer and normal uterine cervix assessed using blood oxygenation level-dependent (BOLD) MRI at 3T†

Hypoxia is reported to be a biomarker for poor prognosis in cervical cancer. However, a practical noninvasive method is needed for the routine clinical evaluation of tumor hypoxia. This study examined the potential use of blood oxygenation level‐dependent (BOLD) contrast MRI as a noninvasive technique to assess tumor vascular oxygenation at 3T. Following Institutional Review Board‐approved informed consent and in compliance with the Health Insurance Portability and Accountability Act, successful results were achieved in nine patients with locally advanced cervical cancer [International Federation of Gynecology and Obstetrics (FIGO) stage IIA to IVA] and three normal volunteers. In the first four patients, dynamic T2*‐weighted MRI was performed in the transaxial plane using a multi‐shot echo planar imaging sequence whilst patients breathed room air followed by oxygen (15 dm3/min). Later, a multi‐echo gradient echo examination was added to provide quantitative R2* measurements. The baseline T2*‐weighted signal intensity was quite stable, but increased to various extents in tumors on initiation of oxygen breathing. The signal in normal uterus increased significantly, whereas that in the iliacus muscle did not change. R2* responded significantly in healthy uterus, cervix and eight cervical tumors. This preliminary study demonstrates that BOLD MRI of cervical cancer at 3T is feasible. However, more patients must be evaluated and followed clinically before any prognostic value can be determined. Copyright

Dual 19F/1H MR Gene Reporter Molecules for in Vivo Detection of β-Galactosidase

Increased emphasis on personalized medicine and novel therapies requires the development of noninvasive strategies for assessing biochemistry in vivo. The detection of enzyme activity and gene expression in vivo is potentially important for the characterization of diseases and gene therapy. Magnetic resonance imaging (MRI) is a particularly promising tool, since it is noninvasive and has no associated radioactivity, yet penetrates deep tissue. We now demonstrate a novel class of dual (1)H/(19)F nuclear magnetic resonance (NMR) lacZ gene reporter molecule to specifically reveal enzyme activity in human tumor xenografts growing in mice. We report the design, synthesis, and characterization of six novel molecules and evaluation of the most effective reporter in mice in vivo. Substrates show a single (19)F NMR signal and exposure to β-galactosidase induces a large (19)F NMR chemical shift response. In the presence of ferric ions, the liberated aglycone generates intense proton MRI T(2) contrast. The dual modality approach allows both the detection of substrate and the imaging of product enhancing the confidence in enzyme detection.

A Noninvasive Tumor Oxygenation Imaging Strategy Using Magnetic Resonance Imaging of Endogenous Blood and Tissue Water

To present a novel imaging strategy for noninvasive measurement of tumor oxygenation using MR imaging of endogenous blood and tissue water.

Dynamic oxygen challenge evaluated by NMR T1 and T2* – insights into tumor oxygenation

There is intense interest in developing non‐invasive prognostic biomarkers of tumor response to therapy, particularly with regard to hypoxia. It has been suggested that oxygen sensitive MRI, notably blood oxygen level‐dependent (BOLD) and tissue oxygen level‐dependent (TOLD) contrast, may provide relevant measurements. This study examined the feasibility of interleaved T2*‐ and T1‐weighted oxygen sensitive MRI, as well as R2* and R1 maps, of rat tumors to assess the relative sensitivity to changes in oxygenation. Investigations used cohorts of Dunning prostate R3327‐AT1 and R3327‐HI tumors, which are reported to exhibit distinct size‐dependent levels of hypoxia and response to hyperoxic gas breathing. Proton MRI R1 and R2* maps were obtained for tumors of anesthetized rats (isoflurane/air) at 4.7 T. Then, interleaved gradient echo T2*‐ and T1‐weighted images were acquired during air breathing and a 10 min challenge with carbogen (95% O2–5% CO2).

Simultaneous measurement of tissue oxygen level‐dependent (TOLD) and blood oxygenation level‐dependent (BOLD) effects in abdominal tissue oxygenation level studies

To assess oxygenation in abdominal organs with magnetic resonance imaging (MRI), a novel approach is presented to simultaneously measure both T1‐ and T2* ‐maps serially during a single dynamic MRI scan in response to an oxygen challenge.

Improving the Evaluation of Alveolar Bone Grafts With Cone Beam Computerized Tomography

Objective Cone Beam computed tomography (CBCT) is used increasingly as a replacement for periapical x-rays when evaluating alveolar bone grafting. The manufacturers standard settings for dental imaging may not, however, represent the optimal settings for evaluating postoperative alveolar bone grafts. We examined the influence of exposure parameters on CBCT image quality to optimize the quality of CBCT images while reducing the radiation dose to the minimum level necessary to obtain adequate images. Methods A defect was created in a cadaver head to simulate an alveolar cleft, and the area was filled with a synthetic material to simulate an alveolar bone graft. Serial CBCT scans were acquired, systematically varying tube voltage and tube current settings from 72 to 96kV and 3 to 12mA. Region of interest analysis was undertaken, and image quality was evaluated by comparing the ratios of native alveolar bone to soft tissue and the ratios of synthetic bone graft to soft tissue and by assessing image noise. Results Twenty-one CBCT data sets were obtained. Reducing tube voltage (kV) resulted in increased contrast ratio between bone and soft tissue and between synthetic bone graft and soft tissue, with maximal contrast at values of 76 kV/11 mA, 72 kV/12 mA, and 72 kV/11 mA. Of these, the setting with lowest image noise was 76 kV/11 mA. This setting also resulted in a radiation dose of less than half of the manufacturers recommended settings for the same scan volume. Conclusions There is potential to improve CBCT image quality significantly while dramatically reducing the radiation dose during postoperative examinations for alveolar bone grafting in patients with cleft lip and palate.

Dynamic facial asymmetry in patients with repaired cleft lip using 4D imaging (video stereophotogrammetry)

BACKGROUND Unilateral cleft lip is a profoundly asymmetrical condition affecting all hard and soft tissue layers from the nose to the upper lip. Although the asymmetry is minimized through cleft lip repair, a degree of asymmetry inevitably persists. Studies investigating asymmetry in patients with cleft lip have used facial measurements, and static 2D and 3D photography. The nose/lip/mouth area, however, is rarely static in our day to day social interactions. METHODS Non-syndromic patients with cleft lip and palate, and a control group without orofacial clefts underwent 50 frames per second 4D imaging while generating facial expressions including smiling and pouting. Key landmarks were tracked throughout the expression, corrected for head movement and a motion path of each landmark was generated. Asymmetry was assessed for both extent of displacement, using Euclidean distances between frames, and the shape of the motion path using Procrustes analysis. RESULTS Twelve patients were compared in each group with an age range from 8 to 18. Comparing the motion path of key landmarks in the upper lip demonstrated statistically significant differences in both the magnitude and shape of motion during smiling and pouting between cleft and non-cleft groups. CONCLUSION Video stereophotogrammetry of the repaired cleft lip demonstrates asymmetry of both the magnitude of motion as well as asymmetry of the path of the motion itself. This may be due to the effect of the scar tissue from the repair, from the abnormal anatomy involved with cleft lip or a combination of the two.