In vivo GABA+ measurement at 1.5T using a PRESS-localized double quantum filter.

A point-resolved spectroscopy (PRESS)-localized double quantum filter was implemented on a 1.5T clinical scanner for the estimation of gamma-amino butyric acid (GABA) concentrations in vivo. Several calibrations were found to be necessary for consistent results to be obtained. The apparent filter yield was approximately 38%; filter strength was sufficient to reduce the singlet metabolite peaks in vivo to below the level of the noise. Metabolite-nulled experiments were performed, which confirmed that significant overlap occurred between macromolecule signals and the GABA resonance at 3.1 ppm. Although the multiplet arm at 2.9 ppm was confirmed to be relatively free of contamination with macromolecules, some contribution from these and from peptides is likely to remain; therefore, the term GABA+ is used. GABA+ concentrations were estimated relative to creatine (Cr) at the same echo time (TE) in a group of controls, studied on two occasions. The GABA+ concentration in 35-ml regions of interest (ROIs) in the occipital lobe was found to be 1.4 +/- 0.2 mM, with scan-rescan repeatability of 38%.

Acute changes in MRI diffusion, perfusion, T(1), and T(2) in a rat model of oligemia produced by partial occlusion of the middle cerebral artery.

Oligemic regions, in which the cerebral blood flow is reduced without impaired energy metabolism, have the potential to evolve toward infarction and remain a target for therapy. The aim of this study was to investigate this oligemic region using various MRI parameters in a rat model of focal oligemia. This model has been designed specifically for remote-controlled occlusion from outside an MRI scanner. Wistar rats underwent remote partial MCAO using an undersize 0.2 mm nylon monofilament with a bullet-shaped tip. Cerebral blood flow (CBF(ASL)), using an arterial spin labeling technique, the apparent diffusion coefficient of water (ADC), and the relaxation times T(1) and T(2) were acquired using an 8.5 T vertical magnet. Following occlusion there was a decrease in CBF(ASL) to 35 +/- 5% of baseline throughout the middle cerebral artery territory. During the entire period of the study there were no observed changes in the ADC. On occlusion, T(2) rapidly decreased in both cortex and basal ganglia and then normalized to the preocclusion values. T(1) values rapidly increased (within approximately 7 min) on occlusion. In conclusion, this study demonstrates the feasibility of partially occluding the middle cerebral artery to produce a large area of oligemia within the MRI scanner. In this region of oligemic flow we detect a rapid increase in T(1) and decrease in T(2). These changes occur before the onset of vasogenic edema. We attribute the acute change in T(2) to increased amounts of deoxyhemoglobin; the mechanisms underlying the change in T(1) require further investigation.

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 relationship between magnetic resonance diffusion imaging and autoradiographic markers of cerebral blood flow and hypoxia in an animal stroke model.

This study examined the relationship between magnetic resonance diffusion imaging and autoradiographic markers of cerebral blood flow (99mTc-hexamethylpropylene amine oxime) and cerebral hypoxia (125I-iodoazomycin arabinoside) in a rat model of stroke. Middle cerebral artery occlusion in the rat was performed using an intraluminal suture approach. Diffusion, hypoxia, and blood flow maps were acquired 2 hr following occlusion, and were compared with T2 images and histology at 7 hr. Two hours following middle cerebral artery occlusion the lesion distributions from the diffusion maps and hypoxic autoradiographs were similar. The blood flow threshold for increased uptake of the hypoxic marker was approximately 34 +/- 7% of the normal flow. The combination of diffusion or hypoxic images with perfusion maps allowed differentiation between four regions: 1) normal tissue; 2) a region of decreased perfusion but normal diffusion and normal uptake of hypoxic marker; 3) a region of decreased perfusion, decreased diffusion and increased uptake of hypoxic marker; 4) a region of decreased perfusion, decreased diffusion and low uptake of hypoxic marker. The areas for increased uptake of hypoxic marker and decreased diffusion are equivalent, indicating similar blood flow thresholds. Regions of oligaemic misery perfusion, ischaemic misery perfusion and lesion core may be delineated with the combination of diffusion or hypoxic images and perfusion maps.

Early changes in water diffusion, perfusion, T1, and T2 during focal cerebral ischemia in the rat studied at 8.5 T.

The time evolution of water diffusion, perfusion, T1, and T2 is investigated at high magnetic field (8.5 T) following permanent middle cerebral artery occlusion in the rat. Cerebral blood flow maps were obtained using arterial spin tagging. Although the quantitative perfusion measurements in ischemic tissue still pose difficulties, the combined perfusion and diffusion data nevertheless distinguish between a "moderately affected area," with reduced perfusion but normal diffusion; and a "severely affected area," in which both perfusion and diffusion are significantly reduced. Two novel magnetic resonance imaging observations are reported, namely, a decrease in T2 and an increase in T1, both within the first few minutes of ischemia. The rapid initial decrease in T2 is believed to be associated with an increase in deoxyhemoglobin levels, while the initial increase in T1 may be related to several factors, such as flow effects, an alteration in tissue oxygenation, and changes in water environment.

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.

Effects of diffusion anisotropy on lesion delineation in a rat model of cerebral ischemia.

The effects of white and gray matter diffusion anisotropy on ischemic lesion delineation have been studied in the rat model of middle cerebral artery occlusion. Apparent diffusion coefficient (ADC) maps obtained by conventional pulsed gradient spin echo diffusion-weighted imaging (PGSE-DWI) were compared with maps of the trace of the diffusion tensor in both normal and occluded animals. Diffusion tensor trace maps were derived from the average of the ADC maps from three separate experiments with diffusion weighting along three orthogonal axes, and also from a single-scan method. A marked degree of diffusion anisotropy was observed in both cortical gray matter and white matter from ADC maps of the control animals. In the occluded animals, the systematic effects of anisotropy on ADC and lesion area influenced the delineation of the ischemic territory in the PGSE-DWI ADC maps. However, the two trace methods eliminated these effects and gave consistent ischemic lesion depiction, despite the use of differing diffusion times in the two measurements.

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.

Proton nuclear magnetic resonance spectroscopy of lactate production in isolated rat liver during cold preservation.

Lactate-edited 1H NMR difference spectra have been acquired from intact rat liver tissue following flushing and preservation in ice. A peak, initially at 1.26 ppm, was seen to increase in the liver tissue with preservation time. This peak was assigned to lactate, despite the fact that its chemical shift was initially shifted by approximately -0.1 ppm relative to an externally added standard. The assignment was based on the following: (a) the peak increased over a 24-h ischemic storage period; (b) it was coupled to a signal 2.78 +/- 0.02 ppm upfield; and (c) a parallel increase in lactate was noted in perchloric acid extracts of tissue from the same liver. An additional peak, assigned to alanine, was also observed during storage and was also shifted by approximately -0.1 ppm. Inclusion of dimethyl sulfoxide, which readily permeates liver tissue, demonstrated that this chemical shift alteration was a tissue-specific effect. These results demonstrate that 1H NMR spectroscopy of intact liver tissue during hypothermic ischemia is possible, though chemical shift assignments should be made with caution.

Metabolic effects of cold storage on livers from euthermic and hibernating Columbian ground squirrels.

The current study was undertaken to investigate energy metabolism during hypoxia in the cold in livers from euthermic and hibernating Columbian ground squirrels. We hypothesized that the hibernating Columbian ground squirrel would be able to maintain liver energetics for a considerably longer time than euthermic animals. Particular reference was made to the function of glycolysis, which is the only mechanism for energy production under hypothermic ischemia. The transition from aerobic to anaerobic metabolism was apparent in both euthermic and hibernating animals as lactate levels rose within 1-3 h; total lactate accumulation was 2.5 micromol/g in both groups. In euthermic squirrels, liver ATP and ADP decreased considerably over the first 3-h storage; values dropped by 55% and 34%, respectively. Conversely, as the drain on high energy phosphate pools progressed, there was an increase in low energy adenylate, AMP. Between 10 and 24 h of storage, increases in AMP accounted for approximately 25-30% of total ATP + ADP decrease. The remainder of the drop in adenylates was accounted for by considerable decreases in total adenylate (TA) contents; by 24 h TA contents had decreased by 2.0 micromol/g. Livers from hibernating squirrels exhibited similar patterns of adenylate change and were not significantly higher than their euthermic counterparts. With respect to regulatory control of glycolysis, livers from euthermic squirrels exhibited no regulatory control at phosphofructokinase (PFK) or pyruvate kinase (PK). Livers from hibernating animals, however, showed an activation at PFK by 10 h of cold storage; levels of hexose phosphates, glucose-6-phosphate + fructose 6-phosphate (G6P + F6P), dropped and fructose 1, 6-biphosphate (F1,6P2), increased. Changes in metabolite levels (phosphoenolpyruvate and pyruvate) associated with another key suspect regulatory enzyme, PK, indicated no role in regulatory control of glycolysis during the 24-h period. The apparent increase in PFK responsiveness to declining energy stores may be a futile activation since there was no accompanying increase in anaerobic end product, lactate, and no maintenance of energetics.

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.

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.

Hypothermic perfusion preservation of liver: the role of phosphate in stimulating ATP synthesis studied by 31P NMR.

Hypothermic perfusion of rat livers was investigated by 31phosphorus nuclear magnetic resonance (31P NMR) spectroscopy using a temperature-controlled module that allowed data acquisition at various time points during a 48-h period. The livers were perfused with an oxygenated lactobionate/raffinose-based solution containing adenosine and inorganic phosphate, and changes in tissue oedema were monitored by direct on-line measurements of liver weight changes. Liver tissue ATP concentrations, determined by fluorimetric assay, were low immediately after organ removal, probably reflecting metabolic stress during the removal period, and these increased slightly during the next 3 h. This was reflected by changes in the 31P NMR spectra. However, by 24 h ATP levels had increased significantly, and these were maintained for up to 48 h, suggesting a shift in the balance between energy production and consumption. When inorganic phosphate was replaced by another anion (citrate), ATP was maintained at a constant lower level during perfusion for 48 h. Tissue weight changes were similar in both groups, suggesting that volume control was not affected by the different ATP contents of the livers. By combining the temperature-controlled module with a separate perfusion circuit, NMR spectroscopy can provide a sensitive method for following energy metabolism in the same organ over long periods during hypothermic perfusion.

Resuscitation of cardiac energy metabolism in the rabbit heart by brief hypothermic reperfusion after preservation studied by 31P NMR spectroscopy.

Rabbit hearts were subjected to 24-h cold ischaemic storage (at 0 degree-2 degrees C in melting ice) after initial flushing with either St Thomas' cardioplegic solution (STS) or modified lactobionate/raffinose solution (LR), and the status of phosphorylated energy metabolites was measured by 31phosphorus nuclear magnetic resonance (P NMR) spectroscopy. In both groups signals for ATP and phosphocreatine (PCr) were still detectable by 31P NMR after 24 h, and there was significantly more ATP in the LR group (P < 0.01). The hearts were then subjected to coronary reperfusion via an aortic cannula using the same storage solution (either STS or LR) at 6 degrees-8 degrees C, which was oxygenated. In both groups PCr recovered within 30 min of cold reperfusion, and by 60 min PCr was significantly higher in the LR group (P < 0.001). Also, levels of ATP were maintained at higher values during cold reperfusion i the LR group. These studies suggest two important points: (1) the general supply of phosphorylated high-energy intermediates of hearts during cold ischaemic storage is better preserved using LR, and (2) brief cold reperfusion may be used to restore energy metabolism in hearts before re-implantation.

Permeation of rabbit common carotid arteries with dimethyl sulfoxide.

Proton nuclear magnetic resonance has been used to measure the kinetics of permeation of dimethyl sulfoxide in the common carotid artery of the rabbit. The process is described by the following exponential equations, where t = time in minutes: % Unexchanged at 2 degrees C = 70e-0.515t + 30e-0.104t and %Unexchanged at 22 degrees C = 70e-1.790t + 30e-0.146t. The times required for 95% equilibration were 13 min at 22 degrees C and 18 min at 2 degrees C. The corresponding times for 99% equilibration were 24 and 32 min, respectively.

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.

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.

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.