Impaired prostate tumorigenesis in Egr1-deficient mice.

The transcription factor early growth response protein 1 (EGR1) is overexpressed in a majority of human prostate cancers and is implicated in the regulation of several genes important for prostate tumor progression. Here we have assessed the effect of Egr1 deficiency on tumor development in two transgenic mouse models of prostate cancer (CR2-T-Ag and TRAMP). Using a combination of high-resolution magnetic resonance imaging and histopathological and survival analyses, we show that tumor progression was significantly impaired in Egr1-/- mice. Tumor initiation and tumor growth rate were not affected by the lack of Egr1; however, Egr1 deficiency significantly delayed the progression from prostatic intra-epithelial neoplasia to invasive carcinoma. These results indicate a unique role for Egr1 in regulating the transition from localized, carcinoma in situ to invasive carcinoma.

Semipermeable Hollow Fiber Phantoms for Development and Validation of Perfusion-Sensitive MR Methods and Signal Models.

Two semipermeable, hollow fiber phantoms for the validation of perfusion-sensitive magnetic resonance methods and signal models are described. Semipermeable hollow fibers harvested from a standard commercial hemodialysis cartridge serve to mimic tissue capillary function. Flow of aqueous media through the fiber lumen is achieved with a laboratory-grade peristaltic pump. Diffusion of water and solute species (e.g., Gd-based contrast agent) occurs across the fiber wall, allowing exchange between the lumen and the extralumenal space. Phantom design attributes include: i) small physical size, ii) easy and low-cost construction, iii) definable compartment volumes, and iv) experimental control over media content and flow rate.

Monitoring the time course of cerebral deoxyglucose metabolism by 31P nuclear magnetic resonance spectroscopy.

The phosphorylation of 2-deoxyglucose by the mammalian brain is used as an index of the brain's energy metabolism. The results of phosphorus-31 nuclear magnetic resonance (31P NMR) monitoring of conscious animals in vivo showed rapid phosphorylation of 2-deoxyglucose by brain tissue. The rate of phosphorylation as determined by 31P NMR was consistent with results achieved by tracer methods using carbon-14-labeled 2-deoxyglucose. However, the disappearance of 2-deoxyglucose-6-phosphate was shown to be faster than that reported by tracer studies and occurred without alterations of intracellular pH and energy homeostasis. These results were confirmed by gas chromatography and mass spectroscopy. It is postulated that 2-deoxyglucose may be metabolized in several ways, including dephosphorylation by a hexose phosphatase.

Improved calibration technique for in vivo proton MRS thermometry for brain temperature measurement.

The most common MR-based approach to noninvasively measure brain temperature relies on the linear relationship between the (1)H MR resonance frequency of tissue water and the tissue's temperature. Herein we provide the most accurate in vivo assessment existing thus far of such a relationship. It was derived by acquiring in vivo MR spectra from a rat brain using a high field (11.74 Tesla [T]) MRI scanner and a single-voxel MR spectroscopy technique based on a LASER pulse sequence. Data were analyzed using three different methods to estimate the (1)H resonance frequencies of water and the metabolites NAA, Cho, and Cr, which are used as temperature-independent internal (frequency) references. Standard modeling of frequency-domain data as composed of resonances characterized by Lorentzian line shapes gave the tightest resonance-frequency versus temperature correlation. An analysis of the uncertainty in temperature estimation has shown that the major limiting factor is an error in estimating the metabolite frequency. For example, for a metabolite resonance linewidth of 8 Hz, signal sampling rate of 2 Hz and SNR of 5, an accuracy of approximately 0.5 degrees C can be achieved at a magnetic field of 3T. For comparison, in the current study conducted at 11.74T, the temperature estimation error was approximately 0.1 degrees C.

Concise derivation of oscillating-gradient-derived ADC.

The apparent diffusion coefficient (ADC) is analyzed for the case of oscillating diffusion-sensitizing gradients in the high-frequency regime. We provide a concise derivation of the analytical expression for the ADC for an arbitrary number of gradient oscillations N and initial phase φ. It is demonstrated that an ultimate goal - to determine the surface-to-volume ratio (S/V) from MR measurements by using oscillating gradients - can be achieved with cosine-type gradients (φ = 0) for an arbitrary N. However, to determine S/V employing gradients with φ ≠ 0 (including the sine-type gradients) and arbitrary N additionally requires prior knowledge of the time-dependent diffusion coefficient D(t). The latter is rarely known a priori but can be estimated under certain limiting conditions: (i) in the short time regime, when the total diffusion time of the measurements, t, is smaller than the characteristic diffusion time of the microstructural system of interest, an analytical expression for D(t) is available (Mitra's expression) and this allows S/V to be determined in the short time regime with sine-type gradients; (ii) in the important case of purely restricted diffusion, D(t) → 0 at sufficiently long time, the signal becomes independent of φ and behaves as for the cosine-type gradients, thus, allowing determination of S/V.

Quantitative determination of tumor blood flow and perfusion via deuterium nuclear magnetic resonance spectroscopy in mice.

Murine RIF-1 tumor blood flow and perfusion were quantified by deuterium NMR using D2O as a freely diffusible tracer. After direct intratumor injection of D2O saline solution, the tracer (HOD) residue from the tumor was detected by deuterium NMR and the deuterium residue washout time course was then analyzed employing multicompartment flow models (S-G. Kim and J.J.H. Ackerman, manuscript submitted for publication). The mean tumor blood flow and perfusion rate was 18.5 +/- 8.5 SD ml/(100 g.min) (n = 46) when analyzed by a two-compartment in-series flow model. A number of tumors (n = 15 out of 61 total) showed a biexponential deuterium tracer washout curve. Application of a three-compartment flow model (S-G. Kim and J.J.H. Ackerman, manuscript submitted for publication) fitted the biexponential residue decay data well and yielded a mean tumor blood flow of 15.7 +/- 9.7 SD, fast- and slow-flow components of 36.8 +/- 19.8 SD and 9.7 +/- 5.8 SD ml/(100 g.min), and a fast-flow component fraction of 21 +/- 10 SD%. Small tumors of less than 0.5 cm3 had faster blood flow, 21.1 +/- 8.4 SD ml/(100 g.min) (n = 27), than large tumors of greater than 1.0 cm3, 9.4 +/- 2.9 SD ml/(100 g.min) (n = 13). The NMR measurement of tumor blood flow and perfusion was not dependent on the number of direct intratumor injection sites and was found reproducible upon repeated measurements of individual tumors. Good agreement with previous in situ photon activation H215O flow determinations was observed.

Nonglycolytic acidification of murine radiation-induced fibrosarcoma 1 tumor via 3-O-methyl-D-glucose monitored by 1H, 2H, 13C, and 31P nuclear magnetic resonance spectroscopy.

The effects of 3-O-methyl-D-glucose (3-OMG) on subcutaneously implanted murine radiation-induced fibrosarcoma 1 tumor were examined with 2H, 13C, and 31P nuclear magnetic resonance (NMR) in situ. Using 31P NMR, changes in tumor high-energy phosphate metabolism were monitored for 2.5 h after i.p. administration of 3-OMG (8.1 g/kg body weight); tumor pH decreased by a mean maximum of 0.52 +/- 0.05 (SE) (n = 10), [PCr] decreased by 54%, [NTP] decreased by 35%, and [Pi] increased by 36%. Tumor blood flow, as measured by 2H NMR monitoring of D2O washout kinetics, decreased by 40% at 1 h and by 47% at 2 h after 3-OMG injection (n = 4). This substantial tumor acidification (pH decrease much greater than 0.1), expected to require a glycolytic substrate (Hwang et al., Cancer Res., 51: 3108-3118, 1991), is surprising in light of the previously documented metabolically inert nature of 3-OMG. In situ 13C NMR spectroscopy, following [6-13C]3-OMG i.p. injection, examined the possibility of the glycolytic metabolism of 3-OMG. However, only the C-6 resonance of 3-OMG was detected (n = 6); no resonances from [6-13C]3-OMG-6-phosphate or [3-13C]lactate were observed. These results confirmed that 3-OMG was not metabolized in radiation-induced fibrosarcoma 1 tumor. At the completion of the in situ 13C NMR experiments, tumors were freeze clamped, and perchloric acid extraction was performed. High-resolution 1H NMR measurement of lactate concentrations showed no statistically significant difference in control tumor extracts (from mice not receiving i.p. injection; n = 5) and in tumor extracts from mice administered i.p. [6-13C]3-OMG (n = 5), indicating that there was no significant increase in lactate level in the tumor extracts from mice administered i.p. 3-OMG due to increased plasma glucose concentration. The results of these 1H and 13C NMR studies indicated that the radiation-induced fibrosarcoma 1 tumor acidification caused by i.p. administration of 3-OMG was not due to a direct (3-OMG----lactate) or an indirect (systemic glucose----lactate) increase in tumor lactic acid levels.

Modulation of murine radiation-induced fibrosarcoma-1 tumor metabolism and blood flow in situ via glucose and mannitol administration monitored by 31P and 2H nuclear magnetic resonance spectroscopy.

The hyperglycemia-induced in situ metabolism and blood flow changes produced in s.c. implanted murine radiation-induced fibrosarcoma-1 tumors, grown on the flanks of female C3H/HeJ mice, were examined with 31P and 2H nuclear magnetic resonance. Initial experiments verified a hyperglycemic tumor acidification similar to that reported earlier with a different substrain of mice, C3H/AnF (J.L. Evelhoch et al., Proc. Natl. Acad. Sci. USA, 81: 6496-6500, 1984). Changes in the tumor pH, phosphorus metabolites, and blood flow were then compared after administration of saline, glucose, or mannitol (a nonmetabolizable glucose analogue) using a mole-equivalent dose of the sugars (i.e., 0.8 mmol/20g mouse). Neither saline (n = 8) nor mannitol (n = 6) administration had any marked effect upon tumor pH, whereas glucose administration produced a mean maximum tumor pH reduction of 0.74 +/- 0.09 (SE; n = 9) during the 2.5 h post-glucose injection. No significant changes in high energy phosphate concentrations were observed during the same period after saline injection. After glucose injection, the [phosphocreatine] gradually decreased by 64% (P = 0.0001). After the initial 1 h post-glucose injection, the [inorganic phosphate] increased by 58% (P = 0.0001), and the [nucleoside triphosphates] decreased by 29% (P = 0.0001) during the following 1.5 h. After mannitol injection, while there was no change in [inorganic phosphate] over time (P = 0.37), the [phosphocreatine] decreased by 33% (P = 0.0001) and the [nucleoside triphosphates] decreased by 21% (P = 0.0015) within 20 min, then both the [phosphocreatine] and [nucleoside triphosphates] remained at constant levels during the following 2 h. In parallel experiments, the volumetric rate of tumor blood flow and perfusion was measured by 2H nuclear magnetic resonance monitoring of 2H2O washout kinetics (S-G. Kim and J. J. H. Ackerman, Cancer Res., 48: 3449-3453, 1988); tumor blood flow decreased by 80% (P = 0.0001, n = 11), 60% (P = 0.0031, n = 4), and 20% (P = 0.058, n = 10) at 2 h after glucose, mannitol, or saline injections, respectively. These results suggest that anaerobic glycolysis is a requirement for hyperglycemic tumor acidification. However, the decrease in tumor blood flow accompanying hyperglycemic acidification suggests that flow reduction also may be a contributing or a required cofactor for acidification via inhibition of lactic acid egress.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct, longitudinal comparison of (1)H and (23)Na MRI after transient focal cerebral ischemia.

BACKGROUND AND PURPOSE: (23)Na MRI may offer new insight into the evaluation of tissue injury. We performed a direct, longitudinal, morphological comparison of (1)H T2 relaxation, (1)H apparent diffusion coefficient (ADC), (23)Na content, and histopathology after cerebral ischemia to address the hypotheses that (a) (23)Na MRI is unique in comparison to (1)H MRI, and (b) accumulation of (23)Na is an unambiguous marker for dead tissue. METHODS: Rats underwent 30 minutes of focal ischemia. MRIs of (1)H T2, (1)H ADC, and (23)Na content were acquired from 12 hours up to 1, 2, or 14 days after reperfusion. On excision, brains were stained with triphenyltetrazolium chloride (TTC). RESULTS: In all cases, the region of abnormality increased in size for 2 days. On day 5, both (1)H T2 and ADC temporarily appeared normal despite the presence of TTC-defined infarction. By comparison, the volume of tissue exhibiting abnormally intense (23)Na signal mirrored the TTC-defined infarct at all time points. CONCLUSIONS: Regions of high (23)Na content correlate well with the TTC-defined infarct and may be a quantitative in vivo marker for dead tissue. In contrast, the dynamics of the (1)H T2 and ADC make it difficult to interpret these images without additional information because they may appear normal despite infarction. Neither type of (1)H image delineates dead tissue, and none of these methods predicts the potential infarct size at early time points.

Correlations between 31P NMR spectroscopy and 15O perfusion measurements in the RIF-1 murine tumor in vivo.

The tumor physiological environment is one of the least understood and most important factors in determining the response of solid tumors to cancer therapy. To examine several important characteristics of the tumor physiological environment we have used in situ photon activation-15O decay measurements (perfusion characteristics) and 31P surface coil-NMR spectroscopy (metabolic characteristics) to observe in vivo subcutaneous RIF-1 tumors grown in female C3H/Anf mice. The following correlations between the 15O perfusion characteristics and the 31P NMR metabolic characteristics in individual tumors were observed: a negative correlation between pH, as measured by NMR (pHNMR), and the inorganic phosphate to nucleosides triphosphate peak height ratio (Pi:NTP); for the well-perfused fraction of the tumor there is a positive correlation with both pHNMR and the phosphocreatine to nucleosides triphosphate peak height ratio (PCr:NTP), and a negative correlation with Pi:NTP. These correlations are interpreted as evidence for a direct relationship between the distribution of cellular physiological environments and the tumor metabolic state. Because these physiological characteristics affect tumor response to various therapeutic modalities and both measurements can be made on humans, it is suggested that these techniques may be of prognostic value in the clinical management of human cancer.

Effects of permeable boundaries on the diffusion-attenuated MR signal: insights from a one-dimensional model.

The local magnetization distribution M(x,t) and the net MR signal S arising from a one-dimensional periodic structure with permeable barriers in a Tanner-Stejskal pulsed-field gradient experiment are considered. In the framework of the narrow pulse approximation, the general expressions for M(x,t) and S as functions of diffusion time and the bipolar field gradient strength are obtained and analyzed. In contrast to a system with impermeable boundaries, the signal S as a function of the b-value is modeled well as a bi-exponential decay not only in the short-time regime but also in the long-time regime. At short diffusion times, the local magnetization M(x,t) is strongly spatially inhomogeneous and the two exponential components describing S have a clear physical interpretation as two "population fractions" of the slow- and fast-diffusing quasi-compartments (pools). In the long-diffusion time regime, the two exponential components do not have clear physical meaning but rather serve to approximate a more complex functional signal form. The average diffusion propagator, obtained by means of standard q-space analysis procedures in the long-diffusion time regime is explored; its structure creates the deceiving appearance of a system with multiple compartments of different sizes, while in reality, it reflects the permeable nature of boundaries in a system with multiple compartments all of the same size.

Transient magnetic resonance without RF pulses: fast field switching.

An unusual strategy for performing magnetic resonance experiments is demonstrated. Instead of employing conventional radiofrequency transmitter fields to perturb spin state populations away from equilibrium, as is the basis of most magnetic resonance spectrometers today, technological advances now make possible fast switching of the magnetic field orientation to achieve the same effect. This is demonstrated with an electron spin resonance experiment where the magnetic field is switched 90 degrees nonadiabatically with a dead time of a few tens of nanoseconds and an electron free induction decay observed.

1H and 2H NMR studies of water in work-free wheat flour doughs.

Proton and deuterium NMR relaxation methods were used to characterize water compartmentalization and hydration in work-free wheat flour doughs. Transverse (spin-spin) relaxation measurements define three motionally unique water compartments in the work-free dough preparations. The apparent occupancy fraction and relative mobility of each water domain are found to be functions of moisture content, temperature, and flour type. Additionally, the number of relaxation-resolved water compartments and their characteristic relaxation rate constants are found to depend critically on both moisture content and the interpulse-delay employed for the multi-pulse relaxation experiments. Under controlled experimental conditions, dynamics between the three water compartments can be observed to be consistent with the onset of flour hydration. The most notable observation during the initial period of hydration is a loss of "free" or "loosely bound" water to environments characterized by less mobility. Freezing studies show that hard wheat doughs have slightly less amorphous, non-freezable water than do soft wheat flour doughs prepared under similar conditions.

Diffusion effects on longitudinal relaxation in poorly mixed compartments.

Diffusion of spins between physical or virtual, communicating compartments having different states of longitudinal magnetization leads to diffusion-driven longitudinal relaxation. Herein, in two model systems, the effects of diffusion-driven longitudinal relaxation are explored experimentally and analyzed quantitatively. In the first case, longitudinal relaxation in a single slice of a water phantom is monitored spectroscopically as a function of slice thickness. In the second case, mimicking vascular flow/diffusion effects, longitudinal relaxation is monitored in a two-compartment, semi-permeable fiber phantom. In both cases, apparent longitudinal relaxation, though clearly multi-exponential, is well-modeled as bi-exponential.

Intracellular water-specific MR of microbead-adherent cells: the HeLa cell intracellular water exchange lifetime.

Quantitative characterization of the intracellular water (1)H MR signal from cultured cells will provide critical biophysical insight into the MR signal from tissues in vivo. Microbeads provide a robust immobilization substrate for the many mammalian cell lines that adhere to surfaces and also provide sufficient cell density for observation of the intracellular water MR signal. However, selective observation of the intracellular water MR signal from perfused, microbead-adherent mammalian cells requires highly effective suppression of the extracellular water MR signal. We describe how high-velocity perfusion of microbead-adherent cells results in short apparent (1)H MR longitudinal and transverse relaxation times for the extracellular water in a thin slice selected orthogonal to the direction of flow. When combined with a spin-echo pulse sequence, this phenomenon provides highly effective suppression of the extracellular water MR signal. This new method is exploited here to quantify the kinetics of water exchange from the intracellular to extracellular spaces of HeLa cells. The time constant describing water exchange from intracellular to extracellular spaces, also known as the exchange lifetime for intracellular water, is 119 +/- 14 ms.

Intracellular water specific MR of microbead-adherent cells: HeLa cell intracellular water diffusion.

The (1)H MR signal arising from flowing extracellular media in a perfused, microbead-adherent cultured cell system can be suppressed with a slice-selective, spin-echo pulse sequence. The signal from intracellular water can, thus, be selectively monitored. Herein, this technique was combined with pulsed field gradients (PFGs) to quantify intracellular water diffusion in HeLa cells. The intracellular water MR diffusion-signal attenuation at various diffusion times was well described by a biophysical model that characterizes the incoherent displacement of intracellular water as a truncated Gaussian distribution of apparent diffusion coefficients (ADCs). At short diffusion times, the water "free" diffusion coefficient and the surface-to-volume ratio of HeLa cells were estimated and were, 2.0 +/- 0.3 microm(2)/ms and 0.48 +/- 0.1 microm(-1) (mean +/- SD), respectively. At long diffusion times, the cell radius of 10.1 +/- 0.4 microm was inferred and was consistent with that measured by optical microscopy. In summary: 1) intracellular water "free" diffusion in HeLa cells was rapid, two-thirds that of pure water; and 2) the cell radius inferred from modeling the incoherent displacement of intracellular water by a truncated Gaussian distribution of ADCs was confirmed by independent optical microscopy measures.

Proton decoupled fluorine nuclear magnetic resonance spectroscopy in situ.

The efficacy of proton decoupling for enhancing the 19F nuclear magnetic resonance (NMR) signal-to-noise ratio and spectral resolution in the intact subject is demonstrated. A geometrically orthogonal cross-coil antenna configuration (Helmholtz pair, surface coil) is employed to provide 40 dB of isolation between the 19F observe and 1H decouple frequencies of 188 and 200 MHz, respectively. Further isolation is achieved through the use of high-quality notch filters on both observation and decoupling channels. Application of 19F-(1H) NMR spectroscopy to the study of 2-fluoro-2-deoxy-D-glucose metabolism in cerebral tissue in situ is presented. Significant improvements in sensitivity and resolution are obtained and result from both a collapse of the JFH multiple structure and a substantial positive nuclear Overhauser effect (NOE). To our knowledge, this is the first such demonstration of 1H decoupling in conjunction with 19F observation for study of the metabolism of a fluorinated compound in the living subject.