Acid Homeostasis Following Partial Ischemia in Neonatal Brain Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

The purpose of this study was to investigate neonatal brain energy metabolism, acid, and lactate homeostasis in the period immediately following partial ischemia. Changes in brain buffering capacity were quantified by measuring mean intracellular brain pH, calculated from the chemical shift of P_i, in response to identical episodes of hypercarbia before and after ischemia. In addition, the relationship between brain buffer base deficit and intracellular pH was compared during and following ischemia. Thus, in vivo ³¹P and ¹H nuclear magnetic resonance spectra were obtained from the brains of seven newborn piglets exposed to sequential episodes of hypercarbia, partial ischemia, and a second episode of hypercarbia in the postischemic recovery period. For the first episode of hypercarbia, brain buffering was similar to values reported for adult animals of other species (percentage pH regulation = 54 ± 16%). During ischemia, the brain base deficit per unit change in pH was ?19 ± 5 mM/pH unit, which is similar to values reported for adult rats. By 20–35 min postischemia, brain acidosis partly resolved in spite of a net increase in lactate concentration. Therefore, the consumption of lactate could not explain acid homeostasis in the first 35 min following ischemia. We conclude that H⁺/HCO^-₃ or other proton equivalent translocation mechanisms must be sufficiently developed in piglet brain to support acid regulation. This is surprising, because a substantial body of evidence implies these processes would be less active in immature brain. The second episode of hypercarbia, from 35 to 65 min postischemia, resulted in a smaller decrease in brain pH compared with the first episode, a result indicating an increase in brain buffering capacity (percentage pH regulation = 79 ± 29%). This was associated with a parallel decrease in brain lactate content, and therefore acid regulation could be attributed to either continued ion translocation or the consumption of lactate. A mild decrease in brain pH and content of energy metabolites was observed, a finding suggesting that the metabolic consequences of severe postischemic hypercarbia are neither particularly dangerous or beneficial.     

Intracellular pH, Lactate, and Energy Metabolism in Neonatal Brain During Partial Ischemia Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Sequential 31P and 1H nuclear magnetic resonance spectra were measured for neonatal piglets (n = 7) to determine the relationship between brain intracellular pH (pHi), lactate, and phosphorylated energy metabolites during partial ischemia. Simultaneous determinations of arterial and cerebral venous blood gases, pH, O2 content, and plasma concentrations of glucose and lactate were also made. Ischemia, induced by bilateral carotid artery ligation plus hemorrhagic hypotension for 35 min, resulted in variable reductions in ATP, phosphocreatine, and increases in Pi, H+, and lactate relative to control levels. In four piglets, whose arterial blood glucose rose above control, brain lactate exceeded 20 mumol g-1 with corresponding decreases in pHi of greater than 0.7 units compared to control levels. The extents of brain acidosis and lactosis showed a strong linear correlation with each other (r = 0.94). Maximal changes in brain lactate, pHi, and ATP at the end of ischemia showed significant positive linear correlations with the control levels of arterial blood glucose, but did not correlate with arterial glucose or arterial cerebral-venous glucose difference values during ischemia. The relationship between pHi and buffer base deficit was comparable to results reported for adult animals up to 20 mumol ml-1. However, in contrast to models proposed for adult brain, the continued linear relationship between pH and higher buffer base levels is most consistent with a theoretical model that assumes the presence of weak acid buffers with pKa values from 6.7 to 5.2.     

Observation of Cerebral Metabolites in an Animal Model of Acute Liver Failure In Vivo: A 1H and 31P Nuclear Magnetic Resonance Study

Acute liver failure was induced in rats by a single intragastric dose of carbon tetrachloride. This causes hepatic centrilobular necrosis, as indicated by histological examinations, and produces a large increase in the activity of serum alanine aminotransferase. The plasma NH4+ level (mean +/- SEM) was 123 +/- 10 microM in the control group and 564 +/- 41 microM in animals with acute liver failure (each n = 5). 31P nuclear magnetic resonance (NMR) was used to monitor brain cortical high-energy phosphate compounds, Pi, and intracellular pH. 1H NMR spectroscopy was utilised to detect additional metabolites, including glutamate, glutamine, and lactate. The results show that the forebrain is capable of maintaining normal phosphorus energy metabolite ratios and intracellular pH despite the metabolic challenge by an elevated blood NH4+ level. There was a significant increase in the brain glutamine level and a concomitant decrease in the glutamate level during hyperammonaemia. The brain lactate level increased twofold in rats with acute liver failure. The results indicate that 1H NMR can be used to detect cerebral metabolic changes in this model of hyperammonaemia, and our observations are discussed in relation to compartmentation of NH4+ metabolism.     

Alterations in Regional Brain Catecholamine Concentrations After Experimental Brain Injury in the Rat

Abstract: Although activation of brain catecholaminergic systems has been implicated in the cerebrovascular and metabolic changes during subarachnoid hemorrhage, cerebral ischemia, cortical ablation, and cortical freeze lesions, little is known of the response of regional brain catecholamine systems to traumatic brain injury. The present study was designed to characterize the temporal changes in concentrations of norepinephrine (NE), dopamine (DA), and epinephrine (E) in discrete brain regions following experimental fluid-percussion traumatic brain injury in rats. Anesthetized rats were subjected to fluid-percussion brain injury of moderate severity (2.2–2.3 atm) and killed at 1 h, 6 h, 24 h, 1 week, and 2 weeks postinjury (n = 6 per timepoint). Control animals (surgery and anesthesia without injury) were killed at identical timepoints (n = 6 per timepoint). Tissue concentrations of NE, DA, and E were evaluated using HPLC. Following brain injury, an acute decrease was observed in DA concentrations in the injured cortex ( p < 0.05) at 1 h postinjury, which persisted up to 2 weeks postinjury. Striatal concentrations of DA were significantly increased ( p < 0.05) only at 6 h postinjury. Hypothalamic concentrations of DA and NE increased significantly beginning at 1 h postinjury ( p < 0.05 and p < 0.05, respectively) and persisted up to 24 h for DA ( p < 0.05) and 1 week ( p < 0.05) for NE. These data suggest that acute alterations occur in regional concentrations of brain catecholamines following brain trauma, which may persist for prolonged periods postinjury.     

Cerebral Acid Buffering Capacity at Different Ages Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Cerebral acidosis occurring during ischemia has been proposed as one determinant of tissue damage. Newborn animals appear to be less susceptible to ischemic tissue damage than adults. One possible component of ischemic tolerance could derive from maturational differences in the extent of acid production and buffering in newborns compared to adults. The purpose of this study was to measure the dependency of acid production on the blood plasma glucose concentrations and acid buffering capacity of piglets at different stages of development. Complete ischemia was induced in 29 piglets ranging in postconceptual age from 111 to 156 days (normal term conception, 115 days). Brain buffering capacity during the first 30 min of ischemia was quantified in vivo, via 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, by measuring the change in intracellular brain pH for a given change in the concentration of compounds that contribute to the production of hydrogen ions. Animals from all four age groups showed a similar linear correlation between preischemia blood glucose concentration and intracellular pH after 30 min of ischemia. For each animal the slope of the plot of intracellular pH versus cerebral buffer base deficit was used to calculate the buffer capacity. Using data obtained over the entire 30 min of ischemia, there was no difference in the mean buffer capacity of the different age groups, nor was there a significant correlation between buffer capacity and age. However, there was a significant increase in buffer capacity for the intracellular pH range 6.6-6.0, compared to 7.0-6.6, for all age groups. No significant differences in buffer capacity for these two pH ranges were observed between any of the age groups. Acid buffering capacity was also measured by performing pH titrations on brain tissue homogenized in the presence of inhibitors of glycolysis and creatine kinase. Plots of homogenate pH versus buffer base deficit showed a nonlinear trend similar to that seen in vivo, indicating an increase in buffer capacity as intracellular pH decreases. A comparison of newborn and 1-month-old brain tissue frozen under control conditions or after 45 min of ischemia revealed no differences that could be attributed to age and a slight decrease in buffer capacity of ischemic brain compared to control brain tissue homogenates. There was no difference between the brain buffering capacity measured in vivo using 31P and 1H NMR and that measured in vitro using brain homogenates.     

Brain Metabolism and Intracellular pH During Ischaemia and Hypoxia: An In Vivo 31P and 1H Nuclear Magnetic Resonance Study in the Lamb

Brain metabolism and intracellular pH were studied during and after episodes of ischaemia and hypoxia-ischaemia in lambs anaesthetised with sodium pentobarbitone. 31P and 1H magnetic resonance spectroscopy methods were used to monitor brain pHi and brain concentrations of Pi, phosphocreatine (PCr), beta--nucleoside triphosphate (beta NTP), and lactate. Simultaneous measurements were made of cerebral blood flow and cerebral oxygen and glucose consumption. Cerebral ischaemia sufficient to reduce oxygen delivery to 75% of control values was associated with a fall in brain pHi and increase in brain Pi. Progressively severe hypoxia-ischaemia was associated with a progressive fall in brain pHi, PCr, and beta NTP and increase in brain Pi. In two animals the increase in brain lactate during hypoxia-ischaemia measured by 1H nuclear magnetic resonance (NMR) could be quantitatively accounted for by the increased net uptake of glucose by the brain in relation to oxygen, but was insufficient to account for the concomitant acidosis according to previous estimates of brain buffering capacity. In four animals brain pHi, PCr, Pi, and beta NTP had returned to normal 1 h after the hypoxic-ischaemic episode. In one animal brain pHi had reverted to normal at a time when 1H NMR indicated persistent elevation of brain lactate.     

Effects of Acute Hyperammonemia on Cerebral Amino Acid Metabolism and pHi In Vivo, Measured by 1H and 31P Nuclear Magnetic Resonance

The effects of an acute intravenous infusion of ammonium acetate on rat cerebral glutamate and glutamine concentrations, energy metabolism, and intracellular pH were measured in vivo with 1H and 31P nuclear magnetic resonance (NMR). The level of blood ammonia maintained by the infusion protocol used in this study (approximately 500 microM, arterial blood) did not cause significant changes in arterial PCO2, PO2, or pH. Cerebral glutamate levels fell to at least 80% of the preinfusion value, whereas glutamine concentrations increased 170% relative to the preinfusion controls. The fall in brain glutamate concentrations followed a time course similar to that of the rise of brain glutamine. There were no detectable changes in the content of phosphocreatine (PCr) or nucleoside triphosphates (NTP), within the brain regions contributing to the sensitive volume of the surface coil, during the ammonia infusion. Intracellular pH, estimated from the chemical shift of the inorganic phosphate resonance relative to the resonance of PCr in the 31P spectrum, was also unchanged during the period of hyperammonemia. 1H spectra, specifically edited to allow quantitation of the brain lactate content, indicated that lactate rose steadily during the ammonia infusion. Detectable increases in brain lactate levels were observed approximately 10 min after the start of the ammonia infusion and by 50 min of infusion had more than doubled. Spectra acquired from rats that received a control infusion of sodium acetate were not different from the spectra acquired prior to the infusion of either ammonium or sodium acetate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal Patterns of Poly(ADP-Ribose) Polymerase Activation in the Cortex Following Experimental Brain Injury in the Rat

The activation of poly(ADP-ribose) polymerase, a DNA base excision repair enzyme, is indicative of DNA damage. This enzyme also undergoes site-specific proteolysis during apoptosis. Because both DNA fragmentation and apoptosis are known to occur following experimental brain injury, we investigated the effect of lateral fluid percussion brain injury on poly(ADP-ribose) polymerase activity and cleavage. Male Sprague-Dawley rats (n = 52) were anesthetized, subjected to fluid percussion brain injury of moderate severity (2.5-2.8 atm), and killed at 30 min, 2 h, 6 h, 24 h, 3 days, or 7 days postinjury. Genomic DNA from injured cortex at 24 h, but not at 30 min, was both fragmented and able to stimulate exogenous poly(ADP-ribose) polymerase. Endogenous poly(ADP-ribose) polymerase activity, however, was enhanced in the injured cortex at 30 min but subsequently returned to baseline levels. Slight fragmentation of poly(ADP-ribose) polymerase was detected in the injured cortex in the first 3 days following injury, but significant cleavage was detected at 7 days postinjury. Taken together, these data suggest that poly(ADP-ribose) polymerase-mediated DNA repair is initiated in the acute posttraumatic period but that subsequent poly(ADP-ribose) polymerase activation does not occur, possibly owing to delayed apoptosis-associated proteolysis, which may impair the repair of damaged DNA.     

Effect of Hypoglycemic Encephalopathy upon Amino Acids, High-Energy Phosphates, and pHi in the Rat Brain In Vivo: Detection by Sequential 1H and 31P NMR Spectroscopy

Metabolic alterations in amino acids, high-energy phosphates, and intracellular pH during and after insulin hypoglycemia in the rat brain was studied in vivo by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Sequential accumulations of 1H and 31P spectra were obtained from a double-tuned surface coil positioned over the exposed skull of a rat while the electroencephalogram was recorded continuously. The transition to EEG silence was accompanied by rapid declines in phosphocreatine, nucleoside triphosphate, and an increase in inorganic orthophosphate in 31P spectra. In 1H spectra acquired during the same time interval, the resonances of glutamate and glutamine decreased in intensity while a progressive increase in aspartate was observed. Following glucose administration, glutamate and aspartate returned to control levels (recovery half-time, 8 min); recovery of glutamine was incomplete. An increase in lactate was detected in the 1H spectrum during recovery but it was not associated with any change in the intracellular pH as assessed in the corresponding 31P spectrum. Phosphocreatine returned to control levels following glucose administration, in contrast to nucleoside triphosphate and inorganic orthophosphate which recovered to only 80% and 200% of their control levels, respectively. These results show that the changes in cerebral amino acids and high-energy phosphates detected by alternating the collection of 1H and 31P spectra allow for a detailed assessment of the metabolic response of the hypoglycemic brain in vivo.     

Regional Levels of Lactate and Norepinephrine After Experimental Brain Injury

Abstract: The recently developed controlled cortical impact model of brain injury in rats may be an excellent tool by which to attempt to understand the neurochemical mechanisms mediating the pathophysiology of traumatic brain injury. In this study, rats were subjected to lateral controlled cortical impact brain injury of low grade severity; their brains were frozen in situ at various times after injury to measure regional levels of lactate, high energy phosphates, and norepinephrine. Tissue lactate concentration in the injury site left cortex was increased in injured animals by sixfold at 30 min and twofold at 2.5 h and 24 h after injury ( p < 0.05). At all postinjury times, lactate concentration was also increased in injured animals by about twofold in the cortex and hippocampus adjacent to the injury site ( p < 0.05). No significant changes occurred in the levels of ATP and phosphocreatine in most of the brain regions of injured animals. However, in the primary site of injury (left cortex), phosphocreatine concentration was decreased by 40% in injured animals at 30 min after injury ( p < 0.05). The norepinephrine concentration was decreased in the injury site left cortex of injured animals by 38% at 30 min, 29% at 2.5 h, and 30% at 24 h after injury ( p < 0.05). The level of norepinephrine was also reduced by ∼20% in the cortex adjacent to the injury site in injured animals. The present results suggest that controlled cortical impact brain injury produces disorder in the neuronal oxidative and norepinephrine metabolism.     

Alterations in Ionized and Total Blood Magnesium After Experimental Traumatic Brain Injury

Experimental evidence suggests that magnesium plays a role in the pathophysiological sequelae of brain injury. The present study examined the variation of blood ionized and total magnesium, as well as potassium, sodium, and ionized calcium, after experimental fluid percussion brain injury in rats. Blood ionized magnesium concentration significantly declined from 0.45 +/- 0.02 to 0.32 +/- 0.02 mM by 30 min postinjury and stayed depressed for the 24-h study period in vehicle-treated rats. Blood total magnesium concentration was 0.59 +/- 0.01 mM and remained stable over time in brain-injured vehicle-treated animals. When magnesium chloride (125 micromol/rat) was administered 1 h postinjury, ionized magnesium levels were restored by 2 h postinjury and remained at normal values up to 24 h following brain trauma. Magnesium treatment also significantly reduced posttraumatic neuromotor impairments 1 and 2 weeks after the insult, but failed to attenuate spatial learning deficits. A significant positive and linear correlation could be established between ionized magnesium levels measured 24 h postinjury and neuromotor outcome at 1 and 2 weeks. We conclude that acute ionized magnesium measurement may be a predictor of long-term neurobehavioral outcome following head injury and that delayed administration of magnesium chloride can restore blood magnesium concentration and attenuate neurological motor deficits in brain-injured rats.     

Brain Metabolism and Intracellular pH During Ischaemia: Effects of Systemic Glucose and Bicarbonate Administration Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy In Vivo in the Lamb

Brain metabolism and intracellular pH were studied during and after episodes of incomplete cerebral ischaemia in lambs under sodium pentobarbitone anaesthesia. 31P and 1H magnetic resonance spectroscopy was used to monitor brain pHi and brain concentrations of inorganic phosphate (Pi), phosphocreatine (PCr), beta-nucleoside triphosphate (beta NTP), and lactate. Simultaneous measurements were made of arterio-cerebral venous concentration differences (AVDs) for oxygen, glucose, and lactate. Cerebral ischaemia was induced by a combination of bilateral carotid clamping and hypotension, and the acute effects of systemic administration of glucose and sodium bicarbonate were examined. The molar ratio of glucose to oxygen uptake by the brain (6G/O2) increased above unity during cerebral ischaemia. Statistically significant AVDs for lactate were not observed. Cerebral ischaemia was associated with a reduction in brain pHi PCr/Pi ratio, and an increase in brain lactate. No effect of arterial plasma glucose on brain lactate concentration or brain pHi was evident during cerebral ischaemia or in the postischaemic period. Administration of sodium bicarbonate systemically in the postischaemic period was associated with a rise in arterial and brain tissue PCO2. A fall in brain pHi occurred which was attributable in part to coincidental brain lactate accumulation. The increase in brain lactate measured by 1H nuclear magnetic resonance in vivo during ischaemia was insufficient to account for the change in buffer base calculated to have occurred from previous estimates of brain buffering capacity.     

Tolerance of Low Intracellular pH During Hypercapnia by Rat Cortical Brain Slices: A 31P/1H NMR Study

Metabolic tolerance of low intracellular pH (pH(i)) was studied in well-oxygenated, perfused, neonatal, rat cerebrocortical brain slices (350 microns thick) by inducing severe hypercapnia. In each of 17 separate experiments 80 brain slices (approximately 3.2 g wet weight) were suspended in an NMR tube, perfused with artificial CSF (ACSF), and studied at 4.7 T with 31P and 1H NMR spectroscopy. Spectra obtained every 5 min monitored relative concentrations of lactate or high-energy phosphate metabolites, from which pH(i) and extracellular pH were determined. Unperturbed slice preparations were metabolically stable for > 10 h, with no significant changes occurring in pHi, ATP, phosphocreatine (PCr), inorganic phosphate, or lactate. Different levels of hypercapnia were produced by sequentially perfusing slices with the following different ACSF batches, each having previously been equilibrated with a specific mixture of CO2 in oxygen: (a) 10% CO2, 15 min of perfusion; (b) 30% CO2, 15 min of perfusion; (c) 50% CO2, 15 min of perfusion; (d) 70% CO2, 30 min of perfusion; (e) 50% CO2, 15 min of perfusion; (f) 30% CO2, 15 min of perfusion; and (g) 10% CO2, 15 min of perfusion. At the completion of this protocol slices were again perfused with fresh ACSF that was equilibrated with a 95% O2/5% CO2 gas mixture. In each of five separate 1H and 31P experiments, brain slices were recovered within 2 h after termination of exposure to high CO2. The pHi was determined from measurements of the chemical shift difference between phosphoethanolamine and PCr, using a calibration curve obtained for our preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional and Temporal Alterations in DNA Fragmentation Factor (DFF)-Like Proteins Following Experimental Brain Trauma in the Rat

DNA fragmentation, an early event in neuronal death following traumatic brain injury, may be triggered by the 40-kDa subunit of DNA fragmentation factor (DFF40). DFF40 is typically bound to the 45-kDa subunit of DFF (DFF45), and activation of DFF40 may occur as a result of caspase-3-mediated cleavage of DFF45 into 30- and 11-kDa fragments. In this study, the intracellular distribution of DFF45 and DFF40 was examined following lateral fluid percussion brain injury of moderate severity (2.4-2.7 atm) in male Sprague-Dawley rats. In the cytosolic fraction (S1) of the injured cortex at 2 and 24 h postinjury, significant decreases in the intensities of DFF45-like proteins at 45- and 32-kDa bands and a concomitant increase in the 11-kDa bands were observed (p < 0.05 vs. uninjured controls). A significant decrease in the intensities of the 32-kDa band in the nuclear (P1) fraction of the injured cortex was observed at 30 min and 2 h postinjury (p < 0.01). Concomitantly, a decrease in DFF40 was observed in the cortical S1 fraction at 2 and 24 h (p < 0.05) and in the P1 fraction at 30 min and 2 h postinjury (p < 0.01). In the hippocampus, DFF45 decreased at 30 min in the P1 and 2 h in the S1 fraction (p < 0.05) and recovered by 24 h postinjury, whereas DFF40 was significantly decreased in the S1 and increased in the P1 fraction at both 2 and 24 h (p < 0.01), which indicated a translocation of DFF40 from cytosol to nucleus. These data are the first to demonstrate that changes in DFF proteins occur after brain trauma and suggest that these changes may play a role in apoptotic cell death via caspase-3-DFF45/DFF40-DNA cleavage observed following traumatic brain injury.     

Phosphorus magnetic resonance spectroscopy 2 h after perinatal cerebral hypoxia-ischemia prognosticates outcome in the newborn piglet

Phosphorus magnetic resonance spectroscopy ((31)P MRS) often reveals apparently normal brain metabolism in the first hours after intrapartum hypoxia-ischemia (HI) at a time when conventional clinical assessment of injury severity is problematic. We aimed to elucidate very-early, injury-severity biomarkers. Twenty-seven newborn piglets underwent cerebral HI: (31)P-MRS measures approximately 2 h after HI were compared between injury groups defined by secondary-energy-failure severity as quantified by the minimum nucleotide triphosphate (NTP) observed after 6 h. For severe and moderate injury versus baseline, [Pi]/[total exchangeable high-energy phosphate pool (EPP)] was increased (p < 0.001 and < 0.02, respectively), and [NTP]/[EPP] decreased (p < 0.03 and < 0.006, respectively): severe-injury [Pi]/[EPP] was also increased versus mild injury (p < 0.04). Mild-injury [phosphocreatine]/[EPP] was increased (p < 0.004). Severe-injury intracellular pH was alkaline versus baseline (p < 0.002). For severe and moderate injury [total Mg]/[ATP] (p < 0.0002 and < 0.02, respectively) and [free Mg] (p < 0.0001 and < 0.02, respectively) were increased versus baseline. [Pi]/[EPP], [phosphocreatine]/[Pi] and [NTP]/[EPP] correlated linearly with injury severity (p < 0.005, < 0.005 and < 0.02, respectively). Increased [Pi]/[EPP], intracellular pH and intracellular Mg approximately 2 h after intrapartum HI may prognosticate severe injury, whereas increased [phosphocreatine]/[EPP] may suggest mild damage. In vivo(31)P MRS may have potential to provide very-early prognosis in neonatal encephalopathy.     

Evaluation of Potential Effectors of Agonal Glycolytic Rate in Developing Brain Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Abstract: Previously we have shown that hypercarbia produces a larger decrease in agonal glycolytic rate in 1-month-old swine than in newborns. In an effort to understand the mechanism responsible for this difference, we tested the hypothesis that hypercarbia produces age-related changes in the concentration of one or more effectors of phosphofructokinase activity. Specifically, in vivo ³¹P and ¹H NMR spectroscopy was used to compare changes in lactate levels, intracellular pH, free magnesium concentration, and content of phosphorylated metabolites for these two age groups at three intervals during the first 1.5 min of complete ischemia in the presence or absence of hypercarbia (P_aco ₂ = 102–106 mm Hg). Hypercarbia produced the same drop in intracellular brain pH for both age groups, but the decrease in phosphocreatine level and increase in inorganic phosphate content were greater in 1-month-olds compared with newborns. During ischemia there was no difference between the magnitude of change in intracellular pH and levels of phosphocreatine and inorganic phosphate in hypercarbic 1-month-olds versus newborns. Under control conditions, i.e., normocarbia and normoxia, the free Mg²⁺ concentration was lower and the fraction of magnesium-free ATP was higher for newborns than 1-month-olds. However, there was no change in these variables for either age group during hypercarbia and early during ischemia. Thus, age-related differences in the relative decrease in agonal glycolytic rate during hypercarbia could not be explained by differences in intracellular pH, inorganic phosphate content, or free magnesium concentration. The [ADP]_free at control was higher in newborns compared with 1-month-olds, and there was no age-related difference in [AMP]_free. These variables did not change for newborns when exposed to hypercarbia, but for 1-month-olds [ADP]_free and [AMP]_free increased during hypercarbia relative to control values. High-energy phosphate utilization during ischemia for hypercarbic 1-month-olds was reduced by 74% compared with normocarbic 1-month-olds during ischemia, whereas the reduction in energy utilization (14%) was not significant for hypercarbic versus normocarbic newborns during ischemia. Because hypercarbia reduces the rate of ATP depletion during ischemia in 1-month-olds to a greater extent than in newborns, the increase in [ADP]_free and [AMP]_free will be slower in the former age group. It follows therefore that for 1-month-olds, the agonal glycolytic rate would not be accelerated by ADP and AMP to the same degree during hypercarbia plus ischemia compared with normocarbic plus ischemia, whereas for newborns hypercarbia has relatively little impact on agonal glycolytic rate.     

Effects of Hypoglycaemia and Hypoxia on the Intracellular pH of Cerebral Tissue as Measured by 31P Nuclear Magnetic Resonance

Changes in high-energy phosphate metabolites and the intracellular pH (pHi) were monitored in cerebral tissue during periods of hypoglycaemia and hypoxia using 31P nuclear magnetic resonance spectroscopy. Superfused brain slices were loaded with deoxyglucose at a concentration shown not to impair cerebral metabolism, and the chemical shift of the resulting 2-deoxyglucose-6-phosphate (DOG6P) peak was used to monitor the pHi. In some experiments with low circulating levels of Pi, the intracellular Pi was visible and indicated a pH identical to that of DOG6P, an observation validating its use as an indicator of pHi in cerebral tissue. The pHi was found to be unchanged during moderate hypoglycaemia; however, mild hypoxia (PO2 = 16.4 kPa) and severe hypoglycaemia produced marked reductions from the normal of 7.2 to 6.8 and 7.0, respectively. Hypoglycaemia caused a fall in the level of both phosphocreatine (PCr) and ATP, whereas hypoxia affected PCr alone, as shown previously. However, the fall in pHi was similar during the two insults, thus indicating that the change in pH is not directly linked to lactate production or to the creatine kinase reaction.     

Local and systemic increase in lipid peroxidation after moderate experimental traumatic brain injury

Traumatic brain injury is a common event associated with neurological dysfunction. Oxidative damage, may contribute to some of these pathologic changes. We used a specific and sensitive marker of lipid peroxidation, the isoprostane 8,12-iso-iPF(2alpha) -VI, to investigate whether local and also systemic lipid peroxidation were induced following lateral fluid percussion (FP) brain injury in the rat. Animals were anesthetized and subjected to lateral FP brain injury of moderate severity, or to sham injury as controls. Urine was collected before anesthesia (baseline), 6 and 24 h after injury. Blood was collected at baseline, 1, 6 and 24 h after injury. Animals were killed 24 h after surgery and their brains removed for biochemical analysis. No significant difference was observed at baseline (preinjury) for urine and plasma 8,12-iso-iPF(2alpha) -VI levels between injured and sham-operated animals. By contrast, plasma and urinary levels increased significantly already at 1 and further increased 24 h following brain injury, when compared to sham-operated animals. Finally, compared with sham, injured animals had a significant increase in brain 8,12-iso-iPF(2alpha) -VI levels. These results demonstrate that moderate brain injury induces widespread brain lipid peroxidation, which is accompanied by a similar increase in urine and plasma. Peripheral measurement of 8,12-iso-iPF(2alpha) -VI levels after brain injury may be a reliable marker of brain oxidative damage.     

Brain Transfer Coefficients for 67Ga: Comparison to 55Fe and Effect of Calcium Deficiency

The transfer coefficients (Kin) for the uptake of gallium-67 (67Ga) into brain and CSF were determined in unanesthetized male Fischer-344 rats fed either a normal or a low-Ca diet. Kin for 67Ga was also compared with transfer coefficients for the uptake of iron-55 (55Fe) and 125I-albumin in control animals. The value of CSF 67Ga Kin was 3 x 10(-7) ml.g-1.s-1 and was 50% larger in low-Ca animals. Brain regional Kin values for 67Ga were 3-9 x 10(-7) ml.g-1.s-1 with no differences in Kin between normal and low-Ca rats. CSF Kin values for 55Fe were 40% and those for albumin were 15% of Kin for 67Ga. For brain, Kin values for 55Fe were 15-40% smaller than for 67Ga, but for albumin the Kin values were 85% less than for 67Ga. 67Ga was found to be 99% bound to plasma proteins, whereas 55Fe was 99.9% bound. The results indicate that metals that are primarily bound to transferrin enter the CSF and brain very slowly. Uptake of both metals was faster than albumin, which may indicate that metal bound to small chelates contributes significantly to brain uptake. In addition, Ca deficiency does not enhance entry of Ga into the brain.     

Localized magnetic resonance spectroscopy measurement of brain lactate during intravenous lactate infusion in healthy volunteers.

Proton magnetic resonance spectroscopy (1H MRS) localized to the left temporal-parietal region in 8 healthy volunteers detected a 2.1-fold +/- 0.7-fold increase (all values +/-SD) in brain lactate during intravenous infusion of 0.5 molar (M) sodium lactate (5 meq/kg over 20 minutes). Significant increases in brain lactate occurred within 5-10 minutes after starting lactate infusion, progressively rose during the infusion, then decreased towards baseline levels during 30 minutes post-infusion. Venous lactate concentration increased from 0.8 +/- 0.2 mM to 10.9 +/- 4.1 mM or 13.6-fold during the infusion. Flow phantom findings in vitro suggest attenuation of 1H MRS blood lactate signal from arteries and veins as a result of flow velocity effects. Correlations between paired blood and brain lactate measurements at each sampling time indicate a non-linear relationship between compartments during lactate infusion.