Ronald J. T. Corbett

Quantitative relationship between brain temperature and energy utilization rate measured in vivo using 31P and 1H magnetic resonance spectroscopy

ABSTRACT: In neonatal and adult animals, modest reduction in brain temperature (2–3°C) during ischemia and hypoxia-ischemia provides partial or complete neuroprotection. One potential mechanism for this effect is a decrease in brain energy utilization rate with consequent preservation of brain ATP, as occurs with profound hypothermia. To determine the extent to which modest hypothermia is associated with a decrease in brain energy utilization rate, in vivo 31P and 1H magnetic resonance spectroscopy (MRS) was used to measure the rate of change in brain concentration of phosphocreatine, nucleoside triphosphate, and lactate after complete ischemia induced by cardiac arrest in 11 piglets (8–16 d). Preischemia metabolite concentrations and MRS-determined rate constants were used to calculate the initial flux of high energy phosphate equivalents (d[∼P]/dt, brain energy utilization rate). Baseline physiologic and MRS measurements were obtained at 38.2°C and repeated after brain temperature was adjusted between 28 and 41°C. This was followed by measurement of d[∼P]/dt during complete ischemia at 1–2°C increments within this temperature range. Adjusting brain temperature did not alter any systemic variable except for heart rate which directly correlated with brain temperature (r = 0.95, p < 0.001). Before ischemia brain temperature inversely correlated with phosphocreatine (r = −0.89, p < 0.001), and reflected changes in the phosphocreatine-ATP equilibrium, because brain temperature inversely correlated with intracellular pH (r = −0.77, p = 0.005). Brain temperature and d[∼P]/dt were directly correlated and described by a linear relationship (slope = 0.61, intercept = −12, r = 0.92, p < 0.001). A reduction in brain temperature from normothermic values of 38.2°C was associated with a decline in d[∼P]/dt of 5.3% per 1°C, and therefore decreases in d[∼P]/dt during modest hypothermia represent a potential mechanism contributing to neuroprotection.

Modest hypothermia provides partial neuroprotection when used for immediate resuscitation after brain ischemia

Intraischemic reduction in temperature of 2-3 °C (modest hypothermia) has been demonstrated to provide partial neuroprotection in neonatal animals. This investigation determined if modest hypothermia initiated immediately after brain ischemia provides neuroprotection. Piglets were studied with rectal temperature maintained during the 1st h after 15 min of brain ischemia at either 38.3 ± 0.3 °C (normothermia, n = 11) or at 35.8± 0.5 °C (modest hypothermia, n = 11). The severity of brain ischemia was similar between groups as indicated by equivalent reduction in mean blood pressure (90 ± 15 to 24 ± 3 versus 92± 13 to 26 ± 3 mm Hg), and changes in cerebral metabolites and intracellular pH (pHi) measured by magnetic resonance spectroscopy(β-nucleoside triphosphate = 44 ± 9 versus 42 ± 18% of control, control = 100%, pHi: 6.25±.15 versus 6.24 ± 0.22 for normothermic and modestly hypothermic groups, respectively). In the first 90 min after ischemia, there were no differences between groups in the duration and extent of brain acidosis, and relative concentrations of phosphorylated metabolites. Categorical assessment of neurobehavior was evaluated at 72 h postischemia (n = 16), or earlier if an animals condition deteriorated (n = 6). Postischemic hypothermia was associated with less severe stages of encephalopathy compared with normothermia (p = 0.05). Histologic neuronal injury was assessed categorically in 16 brain regions, and postischemic hypothermia resulted in less neuronal injury in temporal (p = 0.024) and occipital (p = 0.044) cortex at 10 mm beneath the cortical surface, and in the basal ganglia (p = 0.038) compared with that in normothermia. Modest hypothermia for 1 h immediately after brain ischemia provides partial neuroprotection and may represent an adjunct to resuscitative strategies.

Validation of a Noninvasive Method to Measure Brain Temperature In Vivo Using 1H NMR Spectroscopy

Abstract: The goal of this study was to evaluate the potential of using the difference between the 1H NMR frequencies of water and N‐acetylaspartic acid (NAA) to measure brain temperature noninvasively. All water‐suppressed and non‐water‐suppressed 1H NMR spectra were obtained at a field strength of 4.7 T using a surface coil. Experiments performed on model solutions revealed a decrease in the difference between NMR frequencies for NAA and water as a linear function of increasing temperature from 14 to 45°C. Changing pH in the range 5.5–7.6 produced no discernible trends for concurrent changes in the slope and intercept of the linear relationship. There were minor changes in slope and intercept for solutions containing 80 or 100 mg of protein/ml versus no protein, but these changes were not considered to be of sufficient magnitude to deter the use of this approach to measure brain temperature. The protein content of swine cerebral cortex was found to remain constant from newborn to 1 month old (78 ± 12 mg/g; n = 41). Therefore, data collected for the model solution containing 80 mg of protein/ml were used as a calibration curve to calculate brain temperature in eight swine during control, hypothermia, ischemia, postischemia, or death, over a temperature range of 23–40°C. A plot of 61 temperatures determined from 1H NMR versus temperatures measured from an optical fiber probe sensor implanted 1 cm into the cerebral cortex showed excellent linear agreement (slope = 1.00 ± 0.03, r2 = 0.96). We conclude that 1H NMR spectroscopy presents a practical means of making noninvasive measurements of brain temperature with an accuracy of better than ± 1°C.

The use of the chemical shift of the phosphomonoester P-31 magnetic resonance peak for the determination of intracellular pH in the brains of neonates

The use of the chemical shift of the phosphomonoester P-31 magnetic resonance peak for the determination of intracellular pH has been assessed for piglet and neonatal human brain in vivo. The chemical shift difference between resonance peaks corresponding to phosphoethanolamine and inorganic phosphate, compared with phosphocreatine, was determined for piglets and human neonates. Using in vitro pH titration data to calculate intracellular pH, it was found that pH values from the phosphoethanolamine peak (pH 6.84 to 6.80) were lower than pH estimates from the inorganic phosphate peak (pH 7.22 to 6.99). This difference suggests that phosphoethanolamine and inorganic phosphate may exist in different intracellular environments. Results are presented to demonstrate that the phosphomonoester peak may also be used to measure changes in intracellular pH associated with brain ischemia.

Alterations in Cerebral Blood Flow and Phosphorylated Metabolites in Piglets during and after Partial Ischemia

ABSTRACT: Ventilated piglets were studied before, during (15 min), and after (90 min) hemorrhagic hypotension to correlate a 60% reduction in cerebral blood flow with cerebral energy state using radiolabeled microspheres (n = 12) and in vivo 31P nuclear magnetic resonance spectroscopy (n = 11). Cerebral blood flow (ml · min−1 · 100 g−1) decreased during hypotension (98 ± 28 to 41 ± 28, p < 0.05), increased at 5 min postreperfusion (131 ± 53, p < 0.05), and returned to control values by 90 min postreperfusion. Cerebral O2 uptake was reduced during partial ischemia, remained depressed 5 min postreperfusion, and increased to within 20% of control values at 90 min postreperfusion. Relative to control, hypotension was associated with decreased (p < 0.05) phosphocreatine (62 ± 11%), phosphocreatine/inorganic phosphate ratio (41 ± 10%), and nucleoside triphosphate (82 ± 12%) while inorganic phosphate increased (155 ± 32%, p < 0.05). During ischemia intracellular pH dropped from 7.06 ± 0.07 to 6.59 ± 0.31 (p < 0.05) and the cerebral arteriovenous difference of glucose increased. Phosphorylated metabolites returned to within 10% of control 15 min after blood reinfusion and remained constant thereafter. Based on calculations of ATP synthesis and utilization rates during control and hypotension, we speculate that the rate of energy utilization of the brain during ischemia is reduced 18–49% relative to the control utilization rate.

Glucose-associated alterations in ischemic brain metabolism of neonatal piglets.

Background and Purpose: During global brain ischemia or hypoxia‐ischemia in adults, hyperglycemia is deleterious to the brain. In contrast, similar adverse effects have not been found in neonatal animals. This investigation examined neonatal piglets to determine if there were specific alterations of ischemic brain metabolism associated with different systemic glucose concentrations and to potentially clarify the effects of hyperglycemia during ischemia in neonates. Methods: Two groups of animals (n=12 in each group) were studied during partial ischemia to compare the effects of hyperglycemia (plasma glucose concentration, 258±97 mg% [mean±SD]) with modest hypoglycemia (plasma glucose concentration, 62±23 mg%). A broad spectrum of cerebral blood flow reduction was achieved by combining inflation of a cervical pressure cuff with varying degrees of hemorrhagic hypotension. High‐energy phosphorylated metabolites, intracellular pH, and cerebral blood flow were simultaneously measured using a magnetic resonance spectroscopic technique. Brain metabolic variables (&bgr;‐ATP, inorganic phosphorus, phosphocreatine, intracellular pH) were plotted as a function of blood flow reduction during partial ischemia for each group. Results: During ischemia values of cerebral blood flow were comparably distributed between groups and ranged from 15% to 110% of those of control. At a given reduction of cerebral blood flow, hyperglycemic piglets maintained a higher concentration of &bgr;‐ATP (p=0.011) and had a smaller increase in inorganic phosphorus (p<0.001). At cerebral blood flow <50% of control, the intracellular pH of piglets with modest hypoglycemia during partial ischemia was never reduced to <6.46, whereas intracellular pH fell as low as 5.97 for hyperglycemic animals. Conclusions: ATP preservation may account for the differing effects of glucose during ischemia in neonates compared with adults, provided that the accentuated brain acidosis is not deleterious to neonatal brain tissue. (Stroke 1992;23:1504‐1511)

Simultaneous Measurement of Cerebral Blood Flow and Energy Metabolites in Piglets Using Deuterium and Phosphorus Nuclear Magnetic Resonance

This report demonstrates the feasibility of using deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) spectroscopy to make multiple simultaneous determinations of changes in cerebral blood flow, brain intracellular pH, and phosphorylated metabolites for individual animals. In vivo spectra were obtained from the brains of newborn piglets immediately following an intracarotid bolus injection of deuterium oxide. Experiments were performed at magnetic field strengths of 1.9 T (2H NMR only) or 4.7 T (interleaved 2H and 31P NMR). The rate of clearance of deuterium signal was used to calculate cerebral perfusion rates (CBFdeuterium) during a stable control physiologic state and conditions known to alter blood flow. CBFdeuterium values measured at 1.9 T under conditions of control (normocarbia, normotension), hypercarbia, hypocarbia, and varying degrees of ischemia induced by hypotension showed a significant positive correlation with values measured simultaneously using radiolabeled microspheres (CBFdeuterium = 0.4 × CBFmicrospheres + 8; r = 0.8). Simultaneous interleaved 2H and 31P NMR measurements under control conditions indicate that brain energy metabolites and intracellular pH remained at constant levels during the time course of the administration and clearance of deuterium oxide. Also, brain phosphorylated metabolites and intracellular pH did not differ significantly from their preinjection levels. Under control physiologic conditions, CBFdeuterium varied by ±6% and phosphorylated metabolite levels did not show a significant change with time, as measured from 15 blood flow determinations collected over 4 h. The results indicate that CBFdeuterium determinations have excellent reproducibility and do not affect brain energy metabolite levels. The procedures described here have the potential to bring a novel methodology to bear on investigating the relationship between cerebral perfusion and energy status during conditions such as ischemia or asphyxia.

Use of 31P magnetic resonance spectroscopy to characterize evolving brain damage after perinatal asphyxia

We investigated postasphyxial brain damage with 31P magnetic resonance spectroscopy (MRS) and correlated it with neurologic assessment and standard laboratory evaluation during the first 10 months of life in 1 infant, baby G. We compared these observations to 31P MRS data from 7 healthy term newborns, 1 normal infant examined serially over the first 8.5 months of life, and 5 other term infants following perinatal asphyxia. MRS noninvasively provides biochemical correlates of the evolution of brain damage following perinatal asphyxia and suggests that pH derived from the inorganic phosphate peak may serve as a marker for brain injury.

Age-Related Changes in Swine Brain Creatine Kinase-Catalyzed 31P Exchange Measured In vivo Using 31P NMR Magnetization Transfer

31P exchange rates through the creatine kinase-catalyzed interconversion of phosphocreatine and γ-ATP were measured in a total of 27 miniature swine ranging in age from 5 days preterm to 5 weeks old. A steep increase in the forward rate constant for 31P exchange from phosphocreatine (PCr) to γ-ATP was observed between 2 days preterm and 3 days postterm, with a more gradual increase for older ages. In contrast, the [PCr]/[NTP] ratio measured by in vivo 31P nuclear magnetic resonance (NMR) remained constant throughout this age interval and close to unity. Forward and reverse rate constants and the rate of flux for 31P exchange were equal to each other for both preterm and 5-week-old animals, suggesting that the creatine kinase reaction is near-equilibrium for this span of age. Multifrequency steady-state saturation of Pi and PCr compared to single-frequency saturation of PCr produced the same extent of saturation transfer to γ-ATP, and the saturation of Pi alone had no effect on the γ-ATP 31P NMR signal. These results suggest that even for immature swine brain, creatine kinase activity should be adequate to buffer against changes in [ATP] when there is a mismatch between energy supply and energy demand, during conditions such as ischemia or hypoxia. The results from the present study indicate the unlikelihood that previously reported discrepancies between forward and reverse 32P flux rates in rat brain (Shoubridge et al., FEBS Lett 140:288–292, 1982) were due to neglect of γ-ATP to Pi exchange. If the contribution of nonadenosine triphosphate to the in vivo rat brain 31P NMR signal is accounted for in the calculation of reverse flux and a literature value for rat brain [PCr] is used in the calculation of forward flux, then forward and reverse flux rates are equal.

Energy Reserves and Utilization Rates in Developing Brain Measured in vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Age-related changes in cerebral energy utilization were examined in swine, a species whose maximal rate of development is known to occur in the perinatal period. Interleaved in vivo 31P and 1H nuclear magnetic resonance spectroscopy was used to measure the rates of change in cerebral concentrations of phosphocreatine (PCr), nucleoside triphosphates, and lactate following complete ischemia, induced via cardiac arrest, in a total of 19 newborn, 10-day-old, and 1-month-old piglets. Preischemic concentrations of these three metabolites plus glucose and glycogen were determined in a separate experiment on 12 piglets whose brains were funnel-frozen in situ. The rate constants for the PCr and ATP decline and lactate increase were determined by nonlinear regression fits to the experimental data, assuming first-order kinetics. The rate constants and preischemic metabolite concentrations were used to calculate the initial flux of high-energy phosphate equivalents (∼P), which was used as an estimate of cerebral energy utilization at the point when ischemia was initiated. Cerebral energy utilization equaled 6.5 ± 1.9, 9.5 ± 3.2, and 15.1 ± 3.2 μmol ∼P/g/min in newborn, 10-day-old, and 1-month-old piglets, respectively. Within each age group the energy utilization rate was not altered by hyperglycemia-induced increases in cerebral energy reserves, but during hypoglycemia cerebral energy utilization rates decrease. The slope of ∼P versus time decreased with the duration of ischemia, indicating that cerebral energy utilization rates decrease after the first few minutes of ischemia. Newborn piglets had higher cerebral energy utilization rates compared with literature values for newborn rats and mice. This is consistent with the concept that newborns from a species with a perinatal stage of maximal growth and development will have higher cerebral energy demands compared with newborns from a species such as rodents, whose maximal growth occurs postnatally. However, this conclusion remains tentative because literature cerebral utilization rates estimated from the initial slope of ∼P-versus-time plots tend to underestimate the true rate, since the assumption of continued linearity may not be valid for the interval chosen.