Aging-related changes in brain metabolism are altered by early developmental exposure to diazepam.

To study the long-term effects of prenatal diazepam (DZ) exposure, 31P NMR (nuclear magnetic resonance) spectra and levels of thiobarbituric acid (TBA)-reactive material were measured in the brains of rats from 3 to 26 months of age. In control rats, there were aging-related increases in levels of TBA-reactive material, decreases in intracellular pH (pHi) and alterations in phosphocreatine (PCr) utilization. Prenatal (late gestational) DZ exposure induced lasting, dose-related and age-related alterations in levels of TBA-reactive material and pHi. The results indicate that the prenatal chemical environment can influence cellular metabolism throughout the lifetime of the organism, and that the process of aging can in turn interact with the consequences of prenatal drug exposure.

31P NMR spectroscopy and HbO2 cryospectrophotometry in prediction of tumor radioresistance caused by hypoxia.

The aim of this study was to search for possible relationships between the fraction of radiobiologically hypoxic cells in tumors and their 31P NMR spectral parameters and intracapillary HbO2 saturations. Four different tumor lines, two murine sarcomas (KHT, RIF-1) and two human ovarian carcinoma xenografts (MLS, OWI), were used. When tumor volume increased from about 200 mm3 to about 2000 mm3, hypoxic fraction increased from 12 to 23% for the KHT line, from 0.9 to 1.7% for the RIF-1 line, and from 9 to 28% for the MLS line. The OWI line showed similar hypoxic fractions at 200 (17%) and 2000 mm3 (15%). Tumor bioenergetic status decreased, that is, the inorganic phosphate (Pi) resonance increased and the phosphocreatine (PCr) and nucleoside triphosphate beta (NTP beta) resonances decreased, with increasing tumor volume for the KHT, RIF-1, and MLS lines, whereas the OWI line did not show any changes in the 31P NMR spectral parameters during tumor growth. Similarly, tumor HbO2 saturation status, that is, the fraction of vessels with HbO2 saturation above 30%, decreased with increasing tumor volume for the KHT, RIF-1, and MLS lines, but remained unchanged during tumor growth for the OWI line. Although the data indicated a relationship between hypoxic fraction and tumor bioenergetic status as well as tumor HbO2 saturation status within a specific line during tumor growth, there was no correlation between hypoxic fraction and tumor bioenergetic status or tumor HbO2 saturation status across the four tumor lines. This may have occurred because cell survival time under hypoxic stress as well as fraction of non-clonogenic, but metabolically active hypoxic cells differed among the tumor lines. This indicates that 31P NMR spectroscopy and HbO2 cryospectrophotometry data have to be supplemented with other data to be useful in prediction of tumor radioresistance caused by hypoxia.

Myelin basic protein and magnetic resonance imaging for diagnosing radiation myelopathy.

The identification of radiation myelopathy using biochemical assays and imaging techniques has not previously been accomplished but has clear clinical application. Measurement of myelin basic protein (MBP) in the cerebrospinal fluid (CSF) and visualization of the spinal cord using magnetic resonance imaging (MRI) gives a potentially accurate diagnosis of radiation myelopathy. Female New Zealand white rabbits were irradiated to the thoracic spinal cord with single doses of 15-45 Gy. Animals receiving higher doses (greater than or equal to 22 Gy) generally demonstrated an early paresis (4-8 weeks) that temporarily improved, and then progressed to complete paralysis by 14-18 weeks. MBP levels in the CSF became strikingly elevated to 100-1000 times the normal value. Subsequent, experiments in which rabbits were serially assessed for MBP levels demonstrated a transient elevation, which corresponded to the transient paresis, followed by dramatic elevations concurrent with the onset of paralysis. Magnetic resonance imaging (MRI) of the irradiated spinal cord showed a geographically distinct region of abnormality that corresponded to the radiation field. Histopathology demonstrated demyelination, focal astrocytosis, erythrodiapedesis, and perineuronal edema in the irradiated sections. It appears that MBP levels in the CSF reflect not only radiation-induced myelopathy but also transient demyelination, and that MRI may have the potential to indicate the region of damage.

Magnetic resonance imaging of the rabbit eye. Improved anatomical detail using magnetization transfer contrast.

Proton nuclear magnetic resonance (NMR) imaging previously has been used to examine structure and pathologies of the eye. The present study investigates the use of a saturation-transfer technique, which exploits water-macromolecular proton magnetic interactions, to enhance image contrast in the rabbit eye in vivo. Upon steady-state saturation of the macromolecular-proton magnetization, the water-proton signal intensity will decrease in proportion to the degree of water-macromolecular proton magnetic interaction. NMR images of the eye collected using saturation transfer are shown to have superior contrast compared to conventional NMR imaging techniques, in regard to numerous ocular structures, including the iris, ciliary bodies, muscle, lens, and cornea.

Quantitation of galactosemic cataracts in dogs using magnetization transfer contrast-enhanced magnetic resonance imaging.

PURPOSE: Magnetic resonance imaging (MRI) is becoming increasingly important for the diagnosis and characterization of ocular pathologies. A drawback to this technique is that image contrast between different regions of tissue can be obscured because of the similarity of their nuclear magnetic resonance relaxation parameters. This problem is addressed by magnetization transfer contrast (MTC) enhancement, a MRI technique that generates high-contrast images based on characteristic tissue differences resulting from the interaction of water and macromolecules. The purpose of this study was to investigate the feasibility of using MTC-enhanced imaging to monitor quantitatively the lens changes associated with sugar cataract formation in galactose-fed dogs. METHODS: Male beagles fed a diet containing 30% galactose were periodically examined by MRI for changes in tissue character. Each examination included a gradient recalled echo image (M0), an MTC-enhanced gradient recalled echo image (Ms), a T1 image determined from a one-shot T1 imaging sequence, and a T1-weighted image taken from the raw T1 data. Average values were obtained for several regions of interest and tabulated. These were correlated with cataractous stages visually observed by slit lamp biomicroscopy and retroillumination photography. RESULTS: Enhanced image details of the lens and anterior segment that documented osmotic changes from initial cortical vacuole formation to cortical and nuclear changes associated with advanced sugar cataracts were characterized from measurements of parameters obtained from M0, Ms, T1-weighted, and T1 images. Changes in the cross-sectional areas of lenses during sugar cataract formation also were documented. The magnetic resonance images showed visible changes from the onset of cortical vacuole formation. Region of interest (ROI) analysis of the images showed tissue changes occurring throughout the cataract progression. CONCLUSIONS: The MTC-enhanced MRI technique is well suited to detecting lens changes associated with cataractogenesis. All but the earliest changes were readily apparent from the images with no further analysis. Graphic ROI analysis was able to detect regional changes associated the cataract progression for all degrees of severity. Furthermore, the images demonstrated changes in size and shape that would not be detectable by visual inspection.

Kinetic assessment of manganese using magnetic resonance imaging in the dually perfused human placenta in vitro.

The transfer and distribution of paramagnetic manganese was investigated in the dually perfused human placenta in vitro (using 10, 20, 100 microM Mn with and without 54Mn) using magnetic resonance imaging (MRI) and conventional radiochemical techniques. The human placenta concentrated 54Mn rapidly during the first 15 min of perfusion and by 4 hr was four times greater than the concentrations of Mn in the maternal perfusate, while the concentration of Mn in the fetal perfusate was 25% of the maternal perfusate levels. Within placentae, 45% of the 54Mn was free in the 100,000g supernatant, with 45% in the 1,000g pellet. The magnetic field dependence of proton nuclear spin-lattice relaxation time (T1) in placental tissue supports this Mn binding. Mn primarily affected the MRI partial saturation rather than spin-echo images of the human placenta, which provided for the separation of perfusate contributions from those produced by Mn. The washout of the Mn from the placenta was slow compared with its uptake, as determined by MRI. Thus, Mn was concentrated by the human placenta, but transfer of Mn across the placenta was limited in either direction. These studies also illustrate the opportunity for studies of human placental function using magnetic resonance imaging as a noninvasive biomarker.

Low-flow hypothermic cardiopulmonary bypass protects the brain.

Cerebral protection during surgical procedures necessitating circulatory arrest or low flow remains the factor that most limits the critical time for repair of lesions. In vivo phosphorus-31 nuclear magnetic resonance spectroscopy was used to assess the metabolic state of the brain during circulatory arrest by measuring the concentration of high-energy phosphate compounds and the intracellular pH. The degree of cerebral protection during deep hypothermic cardiopulmonary bypass at low flow rates was compared with that obtained with a period of circulatory arrest interrupted by intermittent systemic perfusion. Sheep were instrumented with cannulas for cardiopulmonary bypass, and a radiofrequency coil was positioned on the skull. Animals were placed in the bore of a 4.7 Tesla magnet, cooled with the aid of cardiopulmonary bypass to 15 degrees C, and had either circulatory arrest (n = 5) or continuous low flow rates of 5 ml/kg/min (n = 6) or 10 ml/kg/min (n = 7) for 2 hours. A fourth group (n = 5) underwent 1 hour of circulatory arrest, systemic reperfusion for 30 minutes, then another hour of circulatory arrest. Both circulatory arrest and a flow rate of 5 ml/kg/min resulted in severe intracellular acidosis and depletion of high-energy phosphates. A flow of 10 ml/kg/min preserved high-energy phosphates and intracellular pH. Therefore deep hypothermia with cardiopulmonary bypass flows as low as 10 ml/kg/min can maintain brain high-energy phosphate concentrations and intracellular pH for 2 hours in sheep, whereas flows of 5 ml/kg/min or intermittent full-flow systemic perfusion between periods of circulatory arrest offers less protection. Previous studies from our laboratory have shown that improvement in nuclear magnetic resonance parameters positively correlates with improved survival and preservation of neurologic function.

Continuous measurement of ATP by 31P-NMR in term human dually perfused placenta in vitro: response to ischemia.

ATP was examined in dually perfused term human placentas by using 31P-nuclear magnetic resonance (NMR) spectroscopy. 31P-NMR spectra were acquired every 30 min starting approximately 30 min after establishing fetal and maternal perfusions, and maternal perfusate samples were obtained to monitor glucose utilization, lactate production, and human chorionic gonadotropin (hCG) and human placental lactogen (hPL) release. In continuous-perfusion experiments, placentas were perfused as long as 10 h. ATP increased and Pi fell after initiation of perfusion. Fetal volume loss was < 2 ml/h, and constant production of hCG, hPL, and lactate as well as constant utilization of glucose were observed. In additional experiments, ischemia was produced by halting maternal and fetal perfusion pumps after a 2-h control period. After 2, 3, or 4 h of ischemia, ATP decreased 46 +/- 17, 51 +/- 5, and 85% of control, respectively. When perfusion was reinitiated, ATP increased and was maintained for the duration of the experiment (an additional 2 h). Recovery of ATP after reperfusion was not paralleled by recovery in glucose utilization, lactate production, or hPL and hCG release. However, during the reperfusion period, fetal pressure was < 70 mmHg and fetal volume loss was < 2 ml/h. These investigations suggest that the dually perfused human placental lobule can maintain ATP for > or = 10 h. Although the perfused human placenta recovers ATP and maintains fetal perfusion volume after ischemia lasting up to 4 h, utilization of glucose, production of lactate, and production and release of hCG and hPL are impaired.

Hyperglycemia increases cerebral intracellular acidosis during circulatory arrest.

Phosphorus 31 nuclear magnetic resonance spectroscopy was used to assess cerebral high-energy phosphate metabolism and intracellular pH in normoglycemic and hyperglycemic sheep during hypothermic circulatory arrest. Two groups of sheep (n = 8 per group) were placed in a 4.7-T magnet and cooled to 15 degrees C using cardiopulmonary bypass. Spectra were acquired before and during circulatory arrest and during reperfusion and rewarming. Intracellular pH and adenosine triphosphate levels decreased during circulatory arrest. Compared with the normoglycemic animals, the hyperglycemic group was significantly more acidotic with the greatest difference observed during the first 20 minutes of reperfusion (6.40 +/- 0.08 versus 6.08 +/- 0.06; p < 0.001). Intracellular pH returned to baseline after 30 minutes of reperfusion in the normoglycemic group but did not reach baseline until 1 hour of reperfusion in the hyperglycemic animals. Adenosine triphosphate levels were significantly higher in the hyperglycemic group during circulatory arrest. Repletion of adenosine triphosphate during reperfusion was similar for both groups. These results support the hypothesis that hyperglycemia during cerebral ischemia drives anaerobic glycolysis and thus leads to increased lactate production and an increase [corrected] in the intracellular acidosis normally associated with ischemia.

Barbiturates impair cerebral metabolism during hypothermic circulatory arrest.

Barbiturates have been used as a method of cerebral protection in patients undergoing open heart operations. Phosphorus 31 nuclear magnetic resonance spectroscopy was used to assess barbiturate-induced alterations in the cerebral tissue energy state during cardiopulmonary bypass, hypothermic circulatory arrest, and subsequent reperfusion. Sheep were positioned in a 4.7-T magnet with a radiofrequency coil over the skull. Nuclear magnetic resonance spectra were obtained at 37 degrees C, during cardiopulmonary bypass before and after drug administration at 37 degrees C and 15 degrees C, throughout a 1-hour period of hypothermic circulatory arrest, and during a 2-hour reperfusion period. A group of animals (n = 8) was administered a bolus of sodium thiopental (40 mg/kg) during bypass at 37 degrees C followed by an infusion of 3.3 mg.kg-1 x min-1 until hypothermic arrest. A control group of animals (n = 8) received no barbiturate. The phosphocreatine/adenosine triphosphate ratio, reflecting tissue energy state, was lower during cardiopulmonary bypass at 15 degrees C in the treated animals compared with controls (1.06 +/- 0.08 versus 1.36 +/- 0.17; p < 0.001). Lower phosphocreatine/adenosine triphosphate ratios were observed throughout all periods of arrest and reperfusion in the barbiturate-treated animals compared with controls (p < or = 0.01). Thiopental prevented the increase in cerebral energy state normally observed with hypothermia and resulted in a decrease in the energy state of the brain during hypothermic circulatory arrest and subsequent reperfusion. These results suggest that thiopental administration before a period of hypothermic circulatory arrest may prove detrimental to the preservation of the energy state of the brain.

Early developmental exposure to benzodiazepine ligands alters brain 31P-NMR spectra in young adult rats.

Alterations in brain high energy phosphate compounds, using 31P-NMR (nuclear magnetic resonance) spectroscopy, were measured in vivo in young adult (3-4 months) rats following prenatal exposure to ligands acting specifically at benzodiazepine (BDZ) binding sites. The exposure induced a decrease in intracellular pH that indicated a predominant interaction of the drugs in utero with central-type BDZ receptor sites. Late gestational exposure to BDZ ligands also induced changes in brain phosphocreatine (PCr) utilization. Exposure to the lowest dose of DZ (1.0 mg/kg) but not the higher dose (2.5 mg/kg) induced a significant change in PCr utilization. Exposure to the central-type BDZ receptor antagonist RO15-1788 alone clearly altered PCr utilization in adult offspring, and DZ (2.5 mg/kg) when administered concurrently was not able to prevent this effect. Though exposure to a peripheral-type ligand (PK11195) had no effect by itself, it converted the effect of the high dose of DZ to that of the low dose. Together, these results indicate an interaction during development between the central and peripheral-type BDZ binding sites on organization and/or regulation of cellular energy metabolism. Normalized ATP levels were not changed by any prenatal treatment indicating adequate buffering of intracellular ATP by phosphocreatine. The dopaminergic antagonist haloperidol did not alter intracellular pH or any index of phosphate metabolism indicating a selective receptor mediated role for BDZ ligands in influences on the long term organization of intracellular phosphate metabolism.

Modeling magnetization transfer using a three-pool model and physically meaningful constraints on the fitting parameters.

A model for water-macromolecular magnetization transfer is presented which addresses the mechanism of coupling between the hydrogen populations and the extraction of physically meaningful parameters from experimental magnetization transfer data. Both physical exchange between bulk-solvent and site-specific hydration-layer hydrogens and intermolecular magnetic dipolar coupling between these specific hydration-layer-solvent and macromolecular hydrogens are explicitly included, leading to a three-pool model for magnetization transfer. It is shown that the three-pool model is well approximated by a two-pool model for coupling between the bulk-solvent and macromolecular hydrogens when the dipolar-coupled solvent hydrogens are a small fraction of the total solvent, and the solvent-macromolecular coupling constant includes both dipolar magnetic, kappa(dip), and physical exchange, kappa(ex), coupling rates. The model is also extended to multiple solvent systems. The model results in a set of coupled equations that predict magnetization transfer spectra as a function of temperature and composition. Physically meaningful constraints on the coupling and relaxation parameters are established for systems in which magnetization transfer has been observed including solvated cross-linked proteins and lipid bilayers. Using parameter estimates based on these constraints, empirical magnetization transfer spectra are well predicted by the model. It is found that the degree of magnetization transfer becomes independent of kappa(dip) and kappa(ex) when these parameters become greater than about 50 s(-1). In the semi-rigid cross-linked protein systems where the mobility of the macromolecular matrix is insensitive to temperature, the magnitude of the observed magnetization transfer is consistent with being limited by the intermolecular dipolar coupling and spin-lattice relaxation in the bulk-solvent phase.

31P NMR spectroscopy in vivo of two murine tumor lines with widely different fractions of radiobiologically hypoxic cells.

Energy and lipid metabolism as well as tumor pH in two murine tumor lines, the KHT and RIF-1 sarcomas, were studied using 31P NMR spectroscopy. Possible relationships between spectral parameters on the one hand and volume fraction of necrosis and fraction of radiobiologically hypoxic cells on the other were investigated. For both tumor lines the PCr and NTP beta resonances decreased and the Pi resonance increased significantly with increasing tumor volume in the volume range 100-4000 mm3. This decrease in bioenergetic status was accompanied by a decrease in tumor pH from about 7.2 to about 6.8. The NTP beta resonance and the tumor pH tended to be somewhat higher and the Pi resonance somewhat lower for the KHT than for the RIF-1 tumors. Linear relationships were found between tumor pH and Pi or (PCr + NTP beta)/Pi for both tumor lines (P much less than 0.05). The PME resonance increased slightly and the PDE resonance decreased slightly during tumor growth and were not significantly different for the KHT and the RIF-1 tumors. The volume fraction of necrosis was about 5 per cent in both lines at a tumor volume of 100 mm3 and increased to about 30 per cent (KHT) and 50 per cent (RIF-1) at a tumor volume of 4000 mm3. The fraction of radiobiologically hypoxic cells was found to increase from 12 to 23 per cent for the KHT line and from 0.9 to 1.7 per cent for the RIF-1 line when tumor volume was increased from about 200 to about 2000 mm3. The volume-dependence of the 31P NMR spectral parameters indicated increased nutritional deprivation and development of hypoxia and necrosis during tumor growth, and was thus qualitatively in good agreement with the changes observed in necrotic and hypoxic fraction. However, quantitative relationships between any spectral parameter and necrotic or hypoxic fraction across tumor lines were not found, implying that other physiological parameters and/or cellular characteristics may contribute significantly to a 31P NMR tumor spectrum. Consequently, 31P NMR spectra of untreated tumors have to be supplemented with other tumor data, e.g. rate of oxygen consumption, cell survival time under hypoxic stress and/or fraction of metabolically active, non-clonogenic hypoxic cells, to be useful in quantitative determination of tumor hypoxia and hence prediction of tumor radioresistance caused by hypoxia.

Magnetic resonance imaging of the galactosemic dog eye using magnetization transfer contrast.

Magnetization transfer contrast (MTC) enhanced magnetic resonance (MR) imaging is a technique that generates high contrast images based on characteristic tissue differences resulting from the interaction of water and macromolecules. In this study, the feasibility of applying this technique to documenting the progression of osmotic sugar cataract formation was investigated in male beagles, initially 6 or 24 month old, fed a diet containing 30% galactose. MTC enhanced magnetic resonance imaging was periodically conducted on these animal's eyes at 2-Tesla. The lens MR images were compared to photographs obtained by photo-slit lamp and retroillumination photography. The MTC technique provided improved image details of the lens and anterior segment that documented osmotic changes from initial cortical vacuole formation to cortical and nuclear changes associated with advanced sugar cataracts. The latter could not be observed by photo-slit lamp or retroillumination photography.

Magnetization transfer contrast in magnetic resonance imaging.

Magnetization transfer contrast (MTC) in magnetic resonance imaging (MRI) is the result of selectively observing the interaction of bulk water protons with the protons contained in macromolecules of a tissue. Since different tissues have different macromolecular compositions, the MTC can generate very high tissue contrast that is based on well-defined physiochemical properties. This is accomplished by combining a saturation transfer technique with standard MRI procedures. The specific practical and theoretical aspects of saturation transfer as it applies to the generation of MTC are reviewed and discussed. In the last 3 years, MTC has been applied to the study of the body, with useful applications demonstrated in evaluating the morphology of the knee joint, eye, brain, breast, and heart. The application of MTC to accentuate MR angiography and contrast agent studies has also been demonstrated. Thus, MTC is becoming another tool towards maximizing the quality and diagnostic potential of MRI. Recent studies on isolated macromolecules have suggested that the MTC effect is specific to the surface chemistry and correlation time of the macromolecules. These latter results indicate that the magnetization transfer process may provide a unique quantitative method of MR tissue characterization based on macromolecule dynamics and chemistry.

Magnetization transfer characterization of hypertensive cardiomyopathy: significance of tissue water content.

Magnetization transfer measurements offer the potential for specific noninvasive tissue characterization. The goal of the present study was to determine if changes in magnetization transfer would accompany the myocardial remodeling that occurs with hypertrophic cardiomyopathy. Using 40-week spontaneously hypertensive rat (SHR) myocardium, T1, T2, and T1 in the presence of off-resonance irradiation (T1sat) were found to be greater compared to Wistar-Kyoto (WKY) controls. The pseudo-first order rate constant of magnetization transfer (kfor) was less in 40-week SHR compared with WKY while the ratio of equilibrium magnetization in the presence and absence of off-resonance irradiation (MsIM0) was not different. The extent to which observed interspecies differences in tissue water content affected these parameters was investigated by dehydrating normal and hypertrophic myocardium. Significant correlations found between tissue water content and T1, T2, T1sat and kfor, but not MsIM0, suggested changes in tissue water dominated the observed interspecies differences in relaxation parameters. Thus, ventricular remodeling in hypertrophic cardiomyopathy does not alter magnetization transfer though the accompanying change in tissue water content influences water proton relaxation.

Quantitative 1H magnetization transfer imaging in vivo.

A major factor contributing to proton (1H) spin-lattice relaxation in biological tissues is believed to be magnetization transfer between 1H in free bulk water and 1H restricted motion associated with macromolecules. We have shown recently that saturation transfer is an effective approach for studying this magnetization transfer process. Herein the determination of magnetization transfer rates in biological tissues is further analyzed by considering the time and power dependencies of saturation transfer. Following these analyses, quantitative magnetization transfer rate constant image maps were collected from the kidney in vivo. These rate constant images may prove useful in quantitative tissue characterization and in the determination of tissue-specific 1H relaxation mechanisms.

Noise reduction in wide-bore magnets using a patient cage.

Significant rf noise reduction is demonstrated by enclosing the patient in a conducting cage grounded to the magnet bore in a 22-cm-bore spectrometer system capable of examining magnetic images and spectroscopy of human limbs. This method of noise reduction was found to be reliable, simple, and efficient for dealing with ambient rf noise in an unshielded room.

Analysis of water-macromolecule proton magnetization transfer in articular cartilage.

These studies were designed to establish which structural elements of cartilage are responsible for proton magnetization transfer between water (Hf) and macromolecules (Hr) observed in MRI studies on articular cartilage. Saturation transfer techniques were used to monitor magnetization transfer in vitro on samples of the two major constituents of cartilage: collagen and proteoglycan. Articular cartilage samples were also evaluated in vitro before and after the removal of the proteoglycan fraction. Isolated hydrated collagen exhibited a significant proton magnetization transfer rate with water. In contrast, proteoglycans exhibited no proton magnetization transfer. Articular cartilage, in vitro, exhibited a high degree of magnetization transfer with water protons consistent with previous MRI studies in vivo. Enzymatic removal of proteoglycan from the cartilage did not alter the magnetization transfer rate between Hr and Hf. These data demonstrate that the structure and concentration of the collagen matrix are the predominant determinants of the magnetization transfer process in articular cartilage with little or no contribution from proteoglycans. This specificity of the magnetization transfer effect may prove useful in the noninvasive evaluation of cartilage composition and structure in vivo.