Edward Proctor

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. II. Changes during 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 ∼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.

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. I. Methodology

CBF has been measured with the hydrogen clearance technique in the two cerebral hemispheres of the gerbil under halothane anaesthesia. This has been correlated with changes in local pH, tissue lactate, and phosphorus energy metabolites measured in the same animals with 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. The NMR measurements were made with two surface coils, one on each hemisphere. This article describes the experimental details and shows that in acute unilateral or bilateral forebrain ischaemia metabolic changes can be monitored by NMR with no significant interhemispheric cross talk. The metabolic effects of reperfusion are also shown. The model allows the definition of the time course of the metabolic consequences of regional ischaemia and reperfusion in individual laboratory animals.

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.

Cell and tissue responses of a murine tumour to phthalocyanine-mediated photodynamic therapy

Mice bearing a subcutaneously growing tumour (Colo 26) were injected intravenously with the photosensitiser chloroaluminum sulphonated phthalocyanine (5 mg/kg) 24 h prior to irradiating the tumour with laser light (675 nm; 50mW, 100 J/tumour). Energy status of the tumour, as assessed by the loss of high energy phosphates in the 31P-nuclear magnetic resonance spectra, was altered dramatically following treatment, such that the ATP fell to undetectable levels within 1 h of light irradiation. However, assessment of the clonogenic capacity of neoplastic cells isolated from dissociated tumours showed that these rapid changes in cellular metabolism were not reflected in similar rapid changes in cell viability. Reductions in clonogenic capacity, which fell to less than 0.1% of control values at 24h postirradiation, closely mirrored those resulting from the cessation of vascular perfusion. Evaluation of tumour blood flow, using the technique of hydrogen washout, showed that the treatment protocol evoked a gradual and selective reduction in flow within the tumour resulting in complete vascular stasis by approximately 5 h after treatment. The results indicate that while chloroaluminum sulphonated phthalocyanine-mediated photodynamic therapy caused early metabolic damage in neoplastic cells, loss of viability paralleled the induction of complete inhibition of vascular flow in the tumour.

31P nuclear magnetic resonance of rat pancreatic grafts.

This study investigates whether phosphate metabolite concentrations and intracellular pH alter in early acute rejection of rat pancreatic allografts. In vitro biochemical assays, in vitro 31P nuclear magnetic resonance spectroscopy, and in vivo 31P NMR spectroscopy of the grafts were compared. Duct-ligated, vascularized rat pancreatic isografts and allografts were transplanted onto the infrarenal aorta of the recipients with inferior vena cava venous drainage. In order to obtain reproducible acute rejection, allografting was performed across a major histocompatibility barrier. For the in vitro experiments freeze-clamped graft extracts were prepared and analyzed for adenosine triphosphate concentration by fluorimetry, then placed in an 8.5 Tesla vertical bore magnet. 31P NMR spectra were recorded using a Bruker AM 360 spectrometer operating at 145.7 MHz for 31P. Spectra were acquired from nontransplanted controls; 3-day, 5-day, and 1-month posttransplant isografts, and 3-day and 5-day posttransplant allografts. All grafts examined were functioning satisfactorily. The ATP content of the extracts was significantly lower in the 3- and 5-day allografts than the respective isografts. Invasive in vivo 31P NMR spectra were recorded using surface coils adjacent to the grafts from functioning 5-day posttransplant isografts and allografts (i.e., 3 days prior to an expected elevation in blood sugar from acute rejection in the allografts). The ATP/inorganic phosphate ratios and pH from the in vivo spectra were significantly lower in the allografts than in the isografts. It is concluded that changes in intracellular metabolism occur early in the process of acute rejection and that 31P NMR spectroscopy may provide a means of diagnosing this before current methods.

Intracellular Metabolites in Rat Muscle following Trauma: A 31P and 1H Nuclear Magnetic Resonance Study

Hind limb skeletal muscle was studied in vivo in a rat trauma model using nuclear magnetic resonance (NMR) spectroscopy. The model used was a 25% body surface area, full-thickness burn administered under anaesthesia. Two groups of six rats were studied. Weight loss was observed in the experimental group whilst the control group continued to gain weight. Concentration ratios involving intramyocellular phosphocreatine (PCr), creatine (Cr), adenosine triphosphate (ATP), inorganic phosphate (Pi), anserine (Ans) and taurine (Tau) were measured. No change in the ratios of PCr/Pi, PCr/ATP, Ans/PCr + Cr and Tau/PCr + Cr were seen between the two groups. Intracellular pH was the same in the two groups. NMR spectroscopy in vivo gives values of Pi and PCr that differ from those obtained by conventional techniques. NMR values are probably more accurate as no degradation occurs during measurement, the measurements being repeatable and noninvasive.

A Guide to the Effective Protection Provided by Hypothermia Against Ischaemic Depolarisation in the Gerbil Brain

With a zero cerebral blood flow (CBF = 0) model of acute ischaemia in the gerbil brain, the development of ischaemic depolarisation (ID) was monitored by direct cortical current. This allowed us to evaluate an on-line, nonsampling scale of the degree of potential neuroprotection conferred by hypothermia in the 20-30°C range. Using the latent time (L) from onset of ischaemia to the resulting ID (the LID), it was found that at CBF = 0, the LID was inversely related to the brain temperature—at 30°C it was 2.7 times greater than at 37°C; 5.5 times at 25°C; and 7.7 times at 20°C. The protection afforded by hypothermia against ID followed a sigmoidal curve with the greatest increase between 25°C and 30°C. LID is a discrete, non-sampling method that can be used in combination with other modalities such as nuclear magnetic resonance to investigate the benefits of hypothermia for neuroprotection in ischaemic brain disease.

Evaluation of Biochemical Processes by NMR

Phosphorus nuclear magnetic resonance (31P NMR) is finding increasing application as a non-invasive method of studying metabolism in living systems. 31P spectra include signals from ATP, phosphocreatine (in muscle and brain), and inorganic phosphate, and in addition to monitoring the relative concentrations of these metabolites, it is also possible to measure the intracellular pH. The technique is therefore ideally suited to examining the changes in high energy phosphates and pH that are associated with periods of ischaemia, anoxia, or hypoxia.