Applications of magnetic resonance spectroscopy and diffusion-weighted imaging to the study of brain biochemistry and pathology.

The first practical demonstration that nuclear magnetic resonance (NMR) spectroscopy could be applied to the study of brain biochemistry in vivo came in 1980, with the studies of the rat brain using a surface coil. Since then the technique has been rapidly and extensively developed into a versatile, non-invasive tool for the investigation of various aspects of brain biochemistry, physiology and disease. NMR is non-destructive and can be used to examine a wide variety of samples, ranging from localized regions within the whole brain in humans or animals, through tissue preparations (perfused organ, tissue slices and homogenates), to isolated cells and aqueous solutions, such as tissue extracts. 31P and 1H NMR spectra deriving from endogenous compounds of the brain in situ allow assessment of tissue metabolites and provide information about high-energy phosphates, lactate, certain amino acids, intracellular pH and ionic concentrations. Exogenous substrates or probes labelled with stable isotopes can also be introduced into the brain and used to monitor metabolism. Animal models of brain diseases have given some impetus to rapid progress in clinical NMR spectroscopy and also magnetic imaging techniques. The purpose of this article is to highlight the type of information available from these NMR techniques, and to present this in a neuroscience context, emphasizing the biochemical, physiological and pathological information that can be obtained using these methods.

Evidence for negative cooperativity in human erythrocyte sugar transport.

1. When D-glucose exchange influx is measure over a wide range of concentrations then two affinity constants (2.27 and 26.0 mM) are evident. This is consistent with a transport model (the allosteric pore model) in which there is negative cooperativity between subunits of the transport protein. 2. The equations for the allosteric pore model interacting with two substrates (or a substrate and an inhibitor) have been derived and have been used to analyse data from exchange inhibition and for mixed infinite-trans uptake experiments. 3. The exchange inhibition of tracer 3-O-methyl-D-glucose, D-xylose and D-fructose uptake by D-glucose also shows evidence for negative cooperativity and for two inhibition constants which are approximately equal to the D-glucose equilibrium exchange affinity constants. 4. The uptake of D-glucose into infinite-trans D-glucose or 3-O-methyl-D-glucose gives Km values of 2.6 and 2.33 mM, respectively. The uptake of 3-O-methyl-D-glucose into infinite-trans D-glucose or 3-O-methyl-D-glucose gives Km values of 6.0 and 4.6 mM, respectively. V values are slightly higher when the internal sugar is 3-O-methyl-D-glucose. 5. In cells that are treated with fluorodinitrobenzene the apparent Ki value for D-glucose inhibition of tracer D-fructose uptake is lowered. It is proposed that this is due to a partially selective effect of FDNB on the internal subunit interface stability constant (the internal pore gate).

Acute cerebral ischaemia: concurrent changes in cerebral blood flow, energy metabolites, pH, and lactate measured with hydrogen clearance and 31P and 1H nuclear magnetic resonance spectroscopy. III. Changes following ischaemia.

CBF has been measured with the hydrogen clearance technique in the two cerebral hemispheres of the gerbil under halothane anaesthesia. At the same time, intracellular pH and the concentrations of lactate and high-energy phosphates were measured in the brain using 1H and 31P nuclear magnetic resonance spectroscopy. Flow and metabolism have been followed during either a 15- or a 30-min ischaemic period (induced by bilateral carotid occlusion) and for up to 1 h of recovery. There was no significant difference between the flow characteristics of the two experimental groups. High-energy phosphate levels and pH returned to control within approximately 20 min of the end of the ischaemic period. Lactate clearance, following a 30-min occlusion, was slower than the recovery of pH. The concentration of free ADP, calculated from the creatine kinase equilibrium, was lower during the recovery phase than under control conditions.

Tissue pH in cold-stored human donor livers preserved in University of Wisconsin solution. A noninavasive clinical study with 31P-magnetic resonance spectroscopy.

It is not known whether the tissue acidosis that accompanies cold storage is the beginning of irreversible cell injury, ultimately leading to cell death, or whether it is a natural "protective" mechanism for cells to survive hypoxic periods. To answer this question, the tissue pH of 45 cold-stored human donor livers preserved in University of Wisconsin solution (UW) was assessed shortly before implantation using noninvasive 31P magnetic resonance spectroscopy. We conclude that tissue pH during cold storage may be partly dependent upon hepatic glycogen stores and donor age. The wide range of tissue pH values that was observed at the time of implantation does not result in significant effects on cellular damage after transplantation. This indicates that tissue pH is not a major determinant for the viability of UW solution-preserved human donor livers, as indicated by postoperative hepatocellular damage and liver synthesis function. The membrane stabilizing and buffering capacity of UW solution appears to protect liver viability against tissue acidosis. Our results also indicate that liver tissue pH can be lower than has been previously assumed in the literature without significant adverse effects on liver viability.

Early changes in cerebral sodium distribution following ischaemia monitored by 23Na magnetic resonance imaging.

23Na magnetic resonance imaging has been used in this preliminary study to investigate early changes in brain sodium signal intensity during and after cerebral ischaemia in a gerbil model. The total sodium signal in selected brain regions decreased between 15 and 30% within 4 min of the onset of ischaemia, and then remained constant throughout the ischaemic period. The same pattern was observed in the eyes. On reperfusion, there was no significant change in the sodium signal over the first 4 min, but by 8 min the signal intensity had returned to or passed through control levels in all regions measured, with the exception of the eyes. These observations are consistent with the loss and resynthesis of ATP as seen in this model, and may be reflecting the redistribution of tissue sodium resulting from energy failure and recovery.

Cryopreservation studies with porcine corneas.

PURPOSE: A new technique for the cryopreservation of rabbit corneas in 20% w/w dimethylsulfoxide, which has been shown to preserve significant structural and functional integrity of the endothelium, was tested in porcine corneas. METHODS: The characteristics of uptake of dimethylsulfoxide into porcine corneas were measured using proton ( 1 H) nuclear magnetic resonance (NMR) spectroscopy. The effect on structural integrity of exposure to 20% w/w dimethylsulfoxide without freezing was first assessed using vital staining (acridine orange and propidium iodide), and optimum temperature conditions for addition and removal of the cryoprotectant were derived. The effects on structural integrity of cryopreservation in 15% and 20% w/w dimethylsulfoxide, and of reducing the degree of cell swelling during cryoprotectant removal following cryopreservation, were then evaluated. RESULTS: The characteristics of uptake of dimethylsulfoxide from a 10% w/w solution fitted a single exponential, resulting in a maximum tissue concentration of 14.6% when the addition occurred on ice, and 18.5% when the addition took place at room temperature. The toxic effects of dimethylsulfoxide in porcine corneas were highly temperature dependent and only evident after removal of the cryoprotectant. Unlike rabbit corneas, cryopreservation of porcine corneas in 15% and 20% w/w dimethylsulfoxide induced substantial endothelial injury which was not improved by reducing the degree of cell swelling that occurred during removal of the cryoprotectant. CONCLUSIONS: Porcine corneas were substantially more susceptible to the toxic effects of dimethyl sulfoxide, and to cryopreservation injury, than rabbit corneas. These results underline the importance of species variation in animal studies aimed at the cryopreservation of human tissue for transplantation.

The application of nuclear magnetic resonance spectroscopy to assess viability in stored tissues and organs.

The use of nuclear magnetic resonance (NMR) spectroscopy to assess metabolic viability in organ preservation is discussed. A brief coverage of the physical principles involved and the biochemical information available from NMR spectroscopy is given. We also present the advantages and disadvantages of the method and outline the future possibilities of the technique in relation to organ preservation.

A sodium magnetic resonance imaging study of acute cerebral ischaemia in the gerbil.

23Na magnetic resonance imaging has been used to investigate sodium changes during and after cerebral ischaemia in a gerbil model. The sodium signal decreased within 4 minutes of the onset of ischaemia, and subsequently increased between 4 and 8 minutes after the onset of reperfusion. These observations may be reflecting the redistribution of tissue sodium resulting from energy failure and recovery.

Determination of the kinetics of permeation of dimethyl sulfoxide in isolated corneas.

Corneal cryopreservation requires that endothelial cells remain viable and intercellular structure be preserved. High viability levels for cryopreserved endothelial cells have been achieved, but preserving intercellular structure, especially endothelial attachment to Descemet's membrane, has proved difficult. Cell detachment apparently is not caused by ice, suggesting osmotic or chemical mechanisms. Knowledge of the permeation kinetics of cryoprotectants (CPAs) into endothelial cells and stroma is essential for controlling osmotic and chemical activity and achieving adequate tissue permeation prior to cooling. Proton nuclear magnetic resonance (NMR) spectroscopy was used to assess the permeation of dimethyl sulfoxide (DMSO) into isolated rabbit corneas. Corneas with intact epithelia were exposed to isotonic medium or 2.0 mol/L DMSO for 60 min and subsequently transferred to 2.0 or 4.0 mol/L DMSO, respectively, at 22, 0, or -10 degrees C. DMSO concentration in the cornea was measured vs time. The Kedem-Katchalsky model was fitted to the data. Hydraulic permeability (m3/N.s) is 7.1 x 10(-13) + 216%-11% at 22 degrees C, 8.2 x 10(-13) + 235%-21% at 0 degree C, and 1.7 x 10(-14) + 19%-16% at -10 degrees C. The reflection coefficient is 1.0 + 2%-1% at 22 degrees C and 0 degree C, and 0.9 +/- 5% at -10 degrees C. Solute mobility (cm/s) is 5.9 x 10(-6) + 6%-11% at 22 degrees C, 3.1 x 10(-6) + 12%-11% at 0 degree C, and 5.0 x 10(-8) cm/s + 59%-40% at -10 degrees C.

The response of liver to lactobionate/raffinose (University of Wisconsin--UW) solution during hypothermic preservation: a study using 31phosphorus nuclear magnetic resonance.

31P nuclear magnetic resonance spectroscopy has been used to study rat livers following flushing with the University of Wisconsin (UW) lactobionate/raffinose solution (N. Jamieson, R. Sundberg, S. Lindell, J. Southard, and F.O. Belzer, Cryobiology 24, 573-574, 1987; M. Kalayoglu, H. Sollinger, R. Stratta, A. D'Alessandro, R. Hoffman, J. Pirsch, and F. O. Belzer, Lancet 1, 617-619, 1988). These studies have revealed that despite the improved storage properties that have been reported for this solution, hepatic ATP and ADP declined at a rate similar to that seen in the more commonly used Marshall's or Collins' solutions. However, there was a significant inhibition of the developing acidosis, such that by 5 hr postflush, the intracellular pH was 7.17 +/- 0.06 (mean +/- SD, n = 5) compared to 6.90 +/- 0.06 for Marshall's solution (4 hr postflush) and 6.94 +/- 0.04 for Collins' solution (4 hr postflush). This did not appear to be due to a buffering effect of the solution, as this was found to be relatively low, but was probably due to a modification of hepatic metabolism caused by the solution itself.

Biochemical consequences of reflushing hypothermically-stored liver with fresh cold perfusate. Studies on rat liver using 31P NMR spectroscopy.

The metabolic response of the rat liver to flushing and reflushing with Marshall's solution at pH 7.2 or pH 7.8 has been studied by 31P nuclear magnetic resonance spectroscopy. The changes in intracellular pH, inorganic phosphate, ATP and phosphomonoesters have been determined from the 31P spectra. We show that the intracellular pH at any stage of the flushing protocol is largely independent of the pH of the medium when using these solutions. However, we demonstrate that there are differences between the efficiency of the two solutions in respect of the rates of hydrolysis of ATP and accumulation of phosphomonoesters. There were also differences in the response of the livers upon reflushing--those livers reflushed at pH 7.2 resynthesized ATP from a lower initial concentration to achieve ATP concentrations similar to those restored in livers reflushed at pH 7.8. These trends were mirrored in the responses of the phosphomonoester peaks (which contain a contribution from AMP). We conclude that short-term control of liver metabolism during hypothermia is possible by use of solutions of different pH, but that for longer-term storage, other approaches may be necessary to maintain metabolic integrity.

Energy metabolism in liver of anoxia-tolerant turtle species (Pseudemys scripta): a model for studying hepatic tolerance to cold hypoxia.

We have studied biochemical markers of energy metabolism and glycolysis by enzyme analyses and 1H NMR spectroscopy in livers of the freshwater turtle, Pseudemys scripta, after vascular flush and cold storage. Values for hepatic ATP content and energy charge remained unchanged for 24 h and showed only small declines between 24 and 48 h of cold hypoxia. Lactate and glucose levels increased over the 48-h period, demonstrating, respectively, progressive glycolysis and glycogenolysis. These observations are in contrast to those made in mammalian liver, where ATP levels fall precipitously during the first few hours of cold hypoxia and glycolysis is inhibited. Additional changes suggested by 1H NMR spectroscopy may indicate a role for other metabolic pathways. Isolated organs of species such as Pseudemys may be useful models for studying the biochemical basis of resistance to cold hypoxic damage.

Metabolic effects of citrate in liver during cold hypoxia studied by 1H NMR spectroscopy.

We propose the use of 1H nuclear magnetic resonance (NMR) spectroscopy to investigate metabolite fluxes in the mammalian liver during cold hypoxia. Rat livers were flushed with one of four different preservation solutions and stored on ice in the same solution. The preservation solutions were: Marshall's hypertonic citrate (HC); carnosine modified HC (HC-C); modified University of Wisconsin (mod UW); and Bretschneider's histidine--typtophan--ketoglutarate (HTK). Liver biopsies were taken before and at 1, 2.5. 4, 24, and 48 h after storage, and freeze-clamped. The liver was extracted with perchloric acid and analyzed by 1H NMR spectroscopy. Components of the individual preservation solutions, such as citrate, histidine, mannitol, and raffinose, were detected in the extracts. Lactate was increased over the first 4 h in all stored livers, but only continued to increase in those stored in HC-C and HTK, reaching significantly high levels of 15 and 14 mumol/g, respectively, by 48 h storage (P < 0.05 and P < 0.01, respectively). Levels of succinate and fumarate in all livers were generally unchanged in the first 0-4 h of storage. However, after 4 h of storage, succinate levels rose in the HC and HC-C livers, while remaining unchanged in mod UW and HTK livers. The presence of citrate in the preservation solutions appeared to enhance the late hepatic synthesis of succinate. Fumarate levels were significantly decreased by 48 h of cold storage, indicating continued fumarate consumption at low temperatures. Despite cold hypoxic conditions, some carbon-substrate cycling appears to continue in mammalian liver via pathways other than glycolysis, and citrate from the preservation solution appears to influence this.

Studies on cryoprotectant equilibration in the intact rat liver using nuclear magnetic resonance spectroscopy: a noninvasive method to assess distribution of dimethyl sulfoxide in tissues.

Nuclear magnetic resonance (NMR) spectroscopy was used in the study of rat livers following flushing with a clinically used preservation solution containing either 12 or 30% (v/v) Me2SO. The extent of equilibration of Me2SO in the tissue after 10-15 min of perfusion with Me2SO and again after subsequent washout with Me2SO-free medium was assessed by 1H NMR spectroscopy. 31P NMR spectroscopy was used to follow the changes in ATP, ADP, inorganic phosphate, and tissue pH. The data show that 1H NMR spectroscopy can be used as a sensitive and rapid method of assessing the equilibration and concentration of compounds such as Me2SO, since these compounds are likely to be present at concentrations greatly in excess of other constituents of the medium and will therefore give rise to strong, easily detected signals. At the same time, 31P NMR spectroscopy can be used to monitor the metabolic status of the tissue reflected in the levels of ATP, ADP, and inorganic phosphate, as well as being a noninvasive monitor of intracellular pH. The possibility of determining the tissue pH in the presence of solutes such as Me2SO is discussed.

Evaluation of cold reperfusion as an indicator of viability in stored organs: a 31P NMR study in rat liver.

Rat livers were studied during hypothermic resuscitation perfusion using 31P nuclear magnetic resonance (NMR) spectroscopy as a viability index. Livers were stored for 48 h after being flushed either with a synthetic solution containing plasma-equivalent concentrations of cations, plus citrate and including gelatin polypeptides as colloid (GC solution), or with a modified lactobionate/raffinose [University of Wisconsin (UWC)] solution. After 48 h in either solution, all NMR-detectable ATP plus ADP had disappeared, inorganic phosphate had increased markedly and pH in the livers had become acidotic. During cold reperfusion, ATP was resynthesized, inorganic phosphate declined, and pH returned toward normal values. ATP recovery and decrease in tissue inorganic phosphate were significantly greater (P < 0.02 and P < 0.005, respectively) after 1 h cold reperfusion with the modified UW compared with reperfusion with the GC. 31P NMR spectroscopy was able to detect differences in the metabolic responses of livers stored in different solutions, and coupled with cold reperfusion may be a useful indicator of viability.