31P-MRS-based determination of brain intracellular and interstitial pH: its application to in vivo H+ compartmentation and cellular regulation during hypoxic/ischemic conditions.

In the last decade, significant progress has been made in the characterization of pH regulation in nervous tissue in vitro. However, little work has been directed at understanding how pH regulatory mechanisms function in vivo. We are interested in how ischemic acidosis can effect pH regulation and modulate the extent of post-ischemic brain damage. We used 31P-MRS to determine normal in vivo pH(i) and pH(e) simultaneously in both the isolated canine brain and the intact rat brain. We observed that the 31P(i) peak in the 31P-MRS spectrum is heterogeneous and can be deconvoluted into a number of discrete constituent peaks. In a series of experiments, we identified these peaks as arising from either extracellular or intracellular sources. In particular, we identified the peak representing the neurons and astrocytes and showed that they maintain different basal pH (6.95 and 7.05, respectively) and behave differently during hypoxic/ischemic episodes.

In vivo microdialysis of 2-deoxyglucose 6-phosphate into brain: a novel method for the measurement of interstitial pH using 31P-NMR.

A unique method for simultaneously measuring interstitial (pHe) as well as intracellular (pHi) pH in the brains of lightly anesthetized rats is described. A 4-mm microdialysis probe was inserted acutely into the right frontal lobe in the center of the area sampled by a surface coil tuned for the collection of 31P-NMR spectra. 2-Deoxyglucose 6-phosphate (2-DG-6-P) was microdialyzed into the rat until a single NMR peak was detected in the phosphomonoester region of the 31P spectrum. pHe and pHi values were calculated from the chemical shift of 2-DG-6-P and inorganic phosphate, respectively, relative to the phosphocreatine peak. The average in vivo pHe was 7.24+/-0.01, whereas the average pHi was 7.05+/-0.01 (n = 7). The average pHe value and the average CSF bicarbonate value (23.5+/-0.1 mEq/L) were used to calculate an interstitial Pco2 of 55 mm Hg. Rats were then subjected to a 15-min period of either hypercapnia, by addition of CO2 (2.5, 5, or 10%) to the ventilator gases, or hypocapnia (PCO2 < 30 mm Hg), by increasing the ventilation rate and volume. pHe responded inversely to arterial Pco2 and was well described (r2 = 0.91) by the Henderson-Hasselbalch equation, assuming a pKa for the bicarbonate buffer system of 6.1 and a solubility coefficient for CO2 of 0.031. This confirms the view that the bicarbonate buffer system is dominant in the interstitial space. pHi responded inversely and linearly to arterial PCO2. The intracellular effect was muted as compared with pHe (slope = -0.0025, r2 = 0.60). pHe and pHi values were also monitored during the first 12 min of ischemia produced by cardiac arrest. pHe decreases more rapidly than pHi during the first 5 min of ischemia. After 12 min of ischemia, pHe and pHi values were not significantly different (6.44+/-0.02 and 6.44+/-0.03, respectively). The limitations, advantages, and future uses of the combined microdialysis/31P-NMR method for measurement of pHe and pHi are discussed.

NMR-based identification of intra- and extracellular compartments of the brain Pi peak.

The Pi peak in a 31P NMR spectrum of the brain can be deconvoluted into six separate Lorentzian peaks with the same linewidth as that of the phosphocreatine peak in the spectrum. In an earlier communication we showed that the six Pi peaks in normal brain represent two extracellular and four intracellular compartments. In that report we have identified the first of the extracellular peaks by marking plasma with infused Pi, thereby substantially increasing the amplitude of the single peak at pH 7.35. 2-Deoxyglucose-6-phosphate (2-DG-6-P) was placed in the brain interstitial space by microdialysis. The resulting 2-DG-6-P peak was deconvoluted into three separate peaks. The chemical shift of the principle 2-DG-6-P peak gave a calculated pH of 7.24 +/- 0.02 for interstitial fluid pH, a value that agreed well with the pH of the second extracellular Pi peak at pH 7.25 +/- 0.01. We identified the intracellular compartments by selectively stressing cellular energy metabolism in three of the four intracellular spaces. A seizure-producing chemical, flurothyl, was used to activate the neuron, thereby causing a demand for energy that could not be completely met by oxidative phosphorylation alone. The resulting loss of high-energy phosphate reserves caused a significant increase in intracellular Pi only in those cells associated with the Pi peak at pH 6.95 +/- 0.01. This suggests that this compartment represents the neuron. Ammonia is detoxified in the astrocyte (glutamine synthetase) by incorporating it into glutamine, a process that requires large amounts of glucose and ATP. The intraarterial infusion of ammonium acetate into the brain stressed astrocyte energy metabolism resulting in an increase in the Pi of the cells at pH of 7.05 +/- 0.01 and 7.15 +/- 0.02. This finding, coupled with our observation that these same cells take up infused Pi probably via the astrocyte end-foot processes, lead us to conclude that these two compartments represent two different types of astrocytes, probably protoplasmic and fibrous, respectively. As a result of this study, we now believe the brain contains four extracellular and four intracellular compartments.

Hyperglycemic damage to mitochondrial membranes during cerebral ischemia: amelioration by platelet-activating factor antagonist BN 50739.

The Pulsinelli-Brierley four-vessel occlusion model was used to study the consequences of hyperglycemic ischemia and reperfusion. Rats were subjected to either 30 min of normo- or hyperglycemic ischemia or 30 min of normo- or hyperglycemic ischemia followed by 60 min of reperfusion. In some animals, 2 mg/kg BN 50739, a platelet-activating factor receptor antagonist, was administered intraarterially either before or after the ischemic insult. The changes in mitochondrial membrane free fatty acid levels, phosphatidylcholine fatty acyl composition, and thiobarbituric acid-reactive material (TBAR) content plus the mitochondrial respiratory control ratio (RCR) were monitored. When the platelet-activating factor antagonist was present during normoglycemia, (a) the mitochondrial free fatty acid release both during and after ischemia was slowed, (b) reacylation of phosphatidylcholine following ischemia was promoted, and (c) TBAR accumulation during and following ischemia was decreased. The detrimental effects of hyperglycemia were muted when BN 50739 was present during ischemia. The RCR was preserved and phosphatidylcholine hydrolysis during ischemia was decreased. TBAR levels were consistently higher in hyperglycemic brain mitochondria both during and after ischemia. The RCR correlated directly with mitochondrial phosphatidylcholine polyunsaturated fatty acid content during ischemia and reperfusion. BN 50739 protection of mitochondrial membranes in brain may be influenced by tissue pH.

NMR studies of Pi-containing extracellular and cytoplasmic compartments in brain.

The inorganic phosphate (Pi) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of Pi. To test the ability of the Marquardt-Levenberg (M-L) nonlinear curve fit algorithm software (Peak-Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated Pi spectra with defined peak centers, areas, and signal-to-noise (S/N) ratios ranging from infinity to 5.8. As the S/N ratio decreased below 15, the M-L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three-compartment model containing a mixture of Pi buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the Pi peaks arising from the three compartments with different pH values if each peak made up 10-35% of total Pi area. In vivo, we identified the plasma compartment by intraarterial infusion of Pi. It was assumed that intracellular compartments contained high-energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that Pi compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2-deoxyglucose (2-DG), 2-DG-6-phosphate (2-DG-6-P) would appear in the PME portion of the spectrum ionized according to pH. Four 2-DG-6-P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger Pi peaks represented intracellular spaces.

Ischemia-induced changes in cerebral mitochondrial free fatty acids, phospholipids, and respiration in the rat.

Changes in the free fatty acid pool size and fatty acyl chain composition of mitochondrial membrane phospholipids and their relation to disruption of mitochondrial function were examined in rat brains after 30 min of cerebral ischemia (Pulsinelli-Brierley model) and 60 min of normoxic reoxygenation. During ischemia, significant hydrolysis of polyunsaturated molecular species from diacyl phosphatidylcholine, particularly fatty acyl 20:4 (arachidonic acid; 20% decrease) and 22:6 (docosahexaenoic acid; 15% decrease), was observed. Thirty minutes of ischemia caused a 16% loss of 18:2 (linoleic acid) from phosphatidylethanolamine. Recirculation for 60 min did not return the polyunsaturated fatty acid content of phospholipids to normal. Total content of free fatty acids increased during ischemia, particularly 18:2 and 22:6, which exhibited the most dramatic rise. The free fatty acid pool size continued to increase during 60 min of recirculation. The respiratory control ratio decreased significantly during 30 min of ischemia with no apparent recovery following 60 min of reoxygenation. The degree of free radical-mediated lipid peroxidation in mitochondria was significantly increased during ischemia and reperfusion. It was concluded that (a) 30 min of cerebral ischemia caused differential degradation in each of the phospholipid classes and preferential hydrolysis of the polyunsaturated molecular species and (b) 60 min of normoxic reperfusion failed to promote reacylation of the mitochondrial phospholipids and restoration of normal respiration.

Effect of the platelet-activating factor antagonist BN 50739 and its diluents on mitochondrial respiration and membrane lipids during and following cerebral ischemia.

Recent evidence suggests that platelet-activating factor plays a role in ischemia-induced neural injury. The Pulsinelli-Brierley four-vessel occlusion model was used to study the effect of a synthetic platelet-activating factor antagonist, BN 50739, and its solvents, either dimethyl sulfoxide or hydroxypropyl-beta-cyclodextrin, on cerebral ischemia-reperfusion. Rats were subjected to either 30 min of ischemia or 30 min of ischemia followed by 60 min of recirculation. Changes in the brain mitochondrial free fatty acid pool size, fatty acyl composition of phospholipids, and respiratory function were monitored. When the BN 50739 (2 mg of BN 50739/kg of body weight i.v.) was administered at the onset of recirculation, it significantly reversed the ischemia-induced accumulation of mitochondrial free fatty acids and loss of polyunsaturated fatty acyl chains from phosphatidylcholine and phosphatidylethanolamine while simultaneously improving mitochondrial respiration. Dimethyl sulfoxide alone decreased the mitochondrial level of malonyldialdehyde and total free fatty acid pool size, but there was no improvement in mitochondrial respiration. Hydroxypropyl-beta-cyclodextrin was reported to be pharmacologically inactive and capable of dissolving BN 50739. However, hydroxypropyl-beta-cyclodextrin alone also caused a significant increase in content of cerebral mitochondrial membrane free fatty acids and hydrolysis of phosphatidylcholine in normoxic control animals. The overall effect of BN 50739 on mitochondrial structure and energy metabolism supports the hypothesis that platelet-activating factor may play a key role in ischemia-induced cerebral injury.

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain.

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p < 0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p < 0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.

Inorganic phosphate compartmentation in the normal isolated canine brain.

In vivo 31P magnetic resonance spectra of 16 isolated dog brains were studied by using a 9.4-T wide-bore superconducting magnet. The observed Pi peak had an irregular shape, which implied that it represented more than one single homogeneous pool of Pi. To evaluate our ability to discriminate between single and multiple peaks and determine peak areas, we designed studies of simulated 31Pi spectra with the signal-to-noise (S/N) ratios ranging from infinity to 4.4 with reference to the simulated Pi peak. For the analysis we used computer programs with a linear prediction algorithm (NMR-Fit) and a Marquardt-Levenberg nonlinear curve-fit algorithm (Peak-Fit). When the simulated data had very high S/N levels, both methods located the peak centers precisely; however, the Marquardt-Levenberg algorithm (M-L algorithm) was the more reliable at low S/N levels. The linear prediction method was poor at determining peak areas; at comparable S/N levels, the M-L algorithm determined all peak areas relatively accurately. Application of the M-L algorithm to the individual experimental in vivo dog brain data resolved the Pi peak into seven or more separate components. A composite spectrum obtained by averaging all spectral data from six of the brains with normal O2 utilization was fitted using the M-L algorithm. The results suggested that there were eight significant peaks with the following chemical shifts: 4.07, 4.29, 4.45, 4.62, 4.75, 4.84, 4.99, and 5.17 parts per million (ppm). Although linear prediction demonstrated the presence of only three peaks, all corresponded to values obtained using the M-L algorithm. The peak indicating a compartment at 5.17 ppm (pH 7.34) was assigned to venous pH on the basis of direct simultaneous electrode-based measurements. On the basis of earlier electrode studies of brain compartmental pH, the peaks at 4.99 ppm (pH 7.16) and 4.84 ppm (pH 7.04) were thought to represent interstitial fluid and the astrocyte cytoplasm, respectively.

Recovery of postischemic brain metabolism and function following treatment with a free radical scavenger and platelet-activating factor antagonists.

We have studied the metabolic and functional effects of two new platelet-activating factor (PAF) antagonists (BN 50726 and BN 50739) and their diluent (dimethyl sulfoxide; DMSO) during reoxygenation of the 14-min ischemic isolated brain. Blood gases, EEG, auditory evoked potentials, cerebral metabolic rate for glucose (CMRglc), and cerebral metabolic rate for oxygen (CMRO2) were monitored throughout the study. Frozen brain samples were taken for measurement of brain tissue high-energy phosphates, carbohydrate content, and thiobarbituric acid-reactive material (TBAR, an indicator of lipid peroxidation) at the end of the study. Following 60 min of reoxygenation in the nontreated 14-min ischemic brains, lactate, AMP, creatine (Cr), intracellular hydrogen ion concentration [H+]i), and TBAR values were significantly higher and ATP, creatine phosphate (PCr), CMRglc, CMRO2, and energy charge (EC) values were significantly lower than the corresponding normoxic control values. PCr and CMRO2 values were significantly higher, and glycogen, AMP, and [H+]i values were significantly lower in the BN 50726-treated ischemic brains than in DMSO-treated ischemic brains. In brains treated with BN 50739, ATP, ADP, PCr, CMRO2, and EC values were significantly higher, and lactate, AMP, Cr, and [H+]i values were significantly lower than corresponding values in the DMSO-treated ischemic brains. TBAR values were near control levels in all brains exposed to DMSO. There was also marked recovery of EEG and auditory evoked potentials in brains treated with DMSO. Treatment with BN 50726 or BN 50739 in DMSO appeared to improve brain mitochondrial function and energy metabolism partly as the result of DMSO action as a free radical scavenger.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute ultrastructural response of hypoxic hypoxia with relative ischemia in the isolated brain.

The acute cortical response to surgical brain isolation and subsequent extracorporal normoxic or 30 min hypoxic (PaO2 = 20 mm Hg) perfusions (hypoxic hypoxia with relative ischemia) was evaluated. Cerebral blood flow, arterial pH and CO2 were maintained constant during both perfusions; only the arterial oxygen content was changed. The isolated brain model used in this and previous investigations produces no qualitative ultrastructural changes in the neocortex following brain isolation and normoxic perfusion. However, the acute cortical structural response to 30 min of hypoxic hypoxia with relative ischemia demonstrated a number of important observations. Hypoxic hypoxia produced ultrastructural responses common to cerebral ischemia such as nuclear chromatin clumping, nucleolar condensation and cytoskeletal breakdown. Although neuronal abnormalities seen after 30 min of hypoxic hypoxia were similar to those acute neuronal changes observed following complete cerebral ischemia without recirculation, they differed three ways: (a) mitochondrial swelling and microvacuolation were observed in many cortical pyramidal neurons. (b) Glycogen particles within astroglial processes were observed even after a 30-min period of hypoxic hypoxia. (c) Perivascular astroglial swelling was minimal despite considerable perineuronal swelling. In contrast, incomplete cerebral ischemia produces mitochondrial changes similar to those in hypoxic hypoxia but also causes the depletion of tissue glycogen and perivascular glial swelling. Thus, hypoxic hypoxia with relative ischemia produces a unique acute ultrastructural response compared to either complete or incomplete cerebral ischemia.

Relating cerebral ischemia and hypoxia to insult intensity.

The contributions of five variables believed to influence the brain's metabolism of O2 during hypoxia [duration, PaO2, delta CMRO2 (the difference between normal and experimental oxygen uptake), O2 availability (blood O2 content.CBF), and O2 deficit (delta CMRO2.duration)] were assessed by stepwise and multiple linear regression. Levels of brain tissue carbohydrates (lactate, glucose, and glycogen) and energy metabolites [ATP, AMP, and creatine phosphate (CrP)] were significantly influenced by O2 deficit during hypoxia, as was final CMRO2. After 60 min of reoxygenation, levels of tissue lactate, glucose, ATP, and AMP were related statistically to the O2 deficit during hypoxia; however, CMRO2 changes were always associated more significantly with O2 availability during hypoxia. Creatine (Cr) and CrP levels in the brain following reoxygenation were correlated more to delta CMRO2 during hypoxia. Changes in some brain carbohydrate (lactate and glucose), energy metabolite (ATP and AMP) levels, and [H+]i induced by complete ischemia were also influenced by O2 deficit. After 60 min of postischemic reoxygenation, brain carbohydrate (lactate, glucose, and glycogen) and energy metabolite (ATP, AMP, CrP, and Cr) correlated with O2 deficit during ischemia. We conclude that "O2 deficit" is an excellent gauge of insult intensity which is related to observed changes in nearly two-thirds of the brain metabolites we studied during and following hypoxia and ischemia.

Evaluation of artificial plasma for maintaining the isolated canine brain.

The use of canine erythrocytes suspended in artificial plasma to maintain the isolated brain was investigated in 18 preparations. Two plasmas were studied: One (AP1) contained electrolytes, amino acids, and albumin; the other (AP2) was similar to CSF and contained a mixture of 37 organic nutrients plus electrolytes and albumin. The CMRO2, CMRglu, and cerebral vascular resistance (CVR) were measured during 2 h of perfusion, and tissue high-energy phosphates were measured at the end of perfusion. The AP1 and AP2 groups were compared with control preparations perfused with canine red blood cells suspended in buffy coat-poor canine plasma. Both CMRO2 and ATP decreased to 60% of the control value; CVR increased to 187% of the control value in both groups following 2 h of perfusion. After 2 h of perfusion, the calculated value of intracellular pH (pHi)--based on creatine kinase equilibrium--remained normal (6.96) for the control brains, but decreased to 6.49 and 6.63, respectively, for the AP1- and AP2-perfused brains. Thus, there appears to be an eventual disruption of normal oxidative metabolism resulting in energy failure, possibly caused by the absence of an essential nutrient from the artificial plasma. For studies of intermediary metabolism in isolated normothermic brain, diluted whole blood appears to be the perfusate of choice.

Cerebral vascular resistance following platelet and leukocyte removal from perfusate.

The rapid increases in cerebral vascular resistance (CVR) frequently observed during extracorporeal perfusion of isolated organs with whole or diluted blood have been investigated using the isolated canine brain preparation. Our data strongly suggest that these increases are caused by a vasoconstrictor that is present in the buffy coat. Plasma serotonin levels were measured and found to be insignificant after conditioning and storage. Conditioning the blood reduced the platelets and the potential for thromboxane A2 production to approximately 40% of normal. However, there was no correlation between thromboxane B2 levels and increases in CVR. Removal of the buffy coat effectively removed all of the leukocytes. Thus, one of the leukotrienes may have been responsible for the buffy coat-induced vasoconstriction.

Vasomotor response to hormonal norepinephrine in canine cerebral and hindlimb vessels.

We calculated the percent change in initial vascular resistance during open loop intra-arterial infusions of norepinephrine (NE) in 12 acutely denervated isolated canine brains and 5 acutely denervated perfused hindlimbs. The studies were repeated on seven chronically denervated brains devoid of perivascular sympathetic fibers. All tissues were free of residual anesthetic. Half-maximal responses were obtained at similar plasma NE concentrations in both the control brains (ED50 13.1 +/- 3.8 X 10(-6) M) and hindlimbs (ED50 9.22 +/- 2.3 X 10(-6). The maximal response was much greater in the limb vasculature (490.3 +/- 30.1%) than in the cerebral vasculature (107.2 +/- 9.5%). A fourfold increase in cerebral vascular sensitivity was noted 3 days after sympathetic gangliectomy (ED50 3.44 +/- 0.74 X 10(-6) M), without any significant change in the maximal response (101.3 +/- 5.7%). Plasma levels of NE observed in resting dogs did not constrict either vascular bed; elevated levels of NE observed in stress dogs would minimally constrict limb vessels but not acute or chronically denervated cerebral vessels. These in situ results confirm that NE does not play a significant role as a physiological vasomotor hormone and suggest that prejunctional neuronal uptake of NE is not responsible for the observed differences in cerebral and hindlimb vascular response. Furthermore, it is unlikely that denervation hypersensitivity to circulating NE plays a role in pathological cerebral vasoconstriction (vasospasm) following subarachnoid hemorrhage.

Cerebral oxygen and energy metabolism during and after 30 minutes of moderate hypoxia.

Sixty-four isolated canine brain preparations were subjected to either 15 or 30 min of perfusion with blood equilibrated at either Pao2 30 mmHg or Pao2 40 mmHg followed by up to 60 min of reoxygenation with blood having a Pao2 greater than 100 mmHg. Pao2 30 mmHg perfusion decreased oxygen availability and the cerebral metabolic rate for oxygen (CMRo2) to 44 and 49% of normal, respectively, whereas Pao2 40 mmHg perfusion decreased oxygen availability and CMRo2 to 64 and 70% of normal, respectively. Creatine phosphate was markedly decreased (0.6 and 4% of normal, respectively) and ATP was only slightly decreased (73 and 90% of normal, respectively) in these preparations during the hypoxic period. Although ATP returned to normal during the reoxygenation period in both groups, creatine phosphate and CMRo2 returned to normal only in the Pao2 40 mmHg preparations. In brains perfused at various Pao2 levels for periods ranging from 6 to 30 min, the total oxygen deficit (the cumulative difference over time between normal and actual CMRo2) rather than tissue lactate levels appeared to influence the restoration of CMRo2 to normal following hypoxia. An oxygen deficit in excess of 25 mumol/g precluded return to a normal CMRo2 following reoxygenation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebral glucose metabolism during 30 minutes of moderate hypoxia and reoxygenation.

In 50 separate experiments, isolated canine brain preparations were subjected to 15 or 30 min of either PaO2 30 mmHg or PaO2 40 mmHg perfusion followed by up to 60 min of reoxygenation at a normal PaO2. The cerebral metabolic rate for glucose (CMRGlu) increased 70-80% after 2 min of hypoxia but then returned to nearly the normal rate by the end of the 30-min period of hypoxia. Glycolytic flux appeared to be facilitated in both groups initially but was inhibited as the hypoxic period continued. This slowing of glycolysis after 15 or 30 min of hypoxia appears to be modulated by the regulatory enzyme phosphofructokinase. Glucose equivalents metabolized, based on CMRGlu plus brain glucose and glycogen disappearance, far exceed the glucose equivalents that can be accounted for on the basis of oxygen utilization and brain lactate formation. Thus, during hypoxia, some of the glucose equivalents must be utilized for synthesis of other metabolites. The glycolytic intermediates returned to normal after reoxygenation in the PaO2 40 mmHg preparations, but the PaO2 30 mmHg preparations continued to show evidence of decreased glycolysis and a lingering lactacidosis. Although posthypoxic oxygen uptake was sufficient to oxidize all glucose entering the brain, there was no significant release of accumulated lactate into the blood. Thus, the decrease in brain tissue lactate must have been the result of lactate oxidation. A significant amount of the glucose entering the brain during the posthypoxic period appears to be used for metabolite synthesis rather than energy production.

Effect of blood pH and ischemia on kinetic constants for cerebral glucose transport.

In 117 experiments, the isolated canine brain was subjected either to 4-min pulses with blood ranging from pH 6.8 to 7.8, 30 min of hypoxia (PaO2 30 mmHg or 40 mmHg), or 30 min of complete ischemia followed by 60 min of perfusion with normal oxygenated blood. Unidirectional and net glucose fluxes were measured under all experimental conditions, and kinetic constants were calculated for unidirectional transport at each pH and after ischemia. In brains perfused with blood having a PaO2 of 30 or 40 mmHg, we observed a 58 and a 55% increase, respectively, in the net flux; however, there was no significant change in the unidirectional flux either during hypoxia or during the recovery period. Exposure of the brains to blood with a pH of 6.8, 7.0, and 7.2 had no effect on the unidirectional flux; however, as pH was raised above 7.4 both the Km and Vmax increased, reaching a maximum of 12.06 +/- 2.34 mM and 2.38 +/- 0.28 mumol X g-1 X min-1, respectively, at pH 7.8. The V/K ratio was unchanged. After 30 min of ischemia, there was a significant change (P less than 0.05) in the Km of the unidirectional glucose transport process from a control value of 5.84 +/- 1.75 mM to 17.40 +/- 5.50. These studies suggest that unidirectional flux is impaired after ischemia due to a decrease in the carrier's affinity for glucose; however, the observed changes are apparently unrelated to a fall in tissue pH. A similar mechanism is believed to be responsible for the decrease in unidirectional glucose flux after hypoxia.

Effects of nitrous oxide on the cerebrovascular tone, oxygen metabolism, and electroencephalogram of the isolated perfused canine brain.

Nitrous oxide has been reported to act both as a stimulant and as a depressant of cerebral oxygen metabolism (CMRO2) and blood flow under a variety of experimental conditions in the intact animal. The isolated brain preparation is advantageous because it permits direct measurement of blood flow and allows the study of drug effects without interference from other organ systems or drugs. In this study, six isolated perfused canine brain preparations were used to compare the CMRO2, cerebral vascular resistance (CVR), and the EEG of brains perfused with normocapnic, normoxic blood equilibrated with either 70% N2O or 70% N2. There was no significant change in CMRO2. Cerebral vascular resistance fell [16.4% +/- 3.4% SEM (P less than 0.015)] during exposure to N2O. The EEG pattern was reduced in amplitude, but showed an increase in both low-voltage beta activity (14-40 Hz), and 3-5 Hz activity. In the isolated brain, N2O reduced cerebral vascular tone while exhibiting no effect on cerebral oxygen metabolism.