Permanent cerebral hypoperfusion: 'preconditioning-like' effects on rat energy metabolism towards acute systemic hypotension

Chronic cerebrovascular disorders are often complicated by additional temporary ischaemic insults, resulting in substantial deterioration of brain energy metabolism. In the present study, chronic limitations of oxygen supply were induced in Wistar rats by 2 weeks of permanent bilateral common carotid artery occlusion (2-vo) to initiate a 'preconditioning-like' effect that protects rat brain energy metabolism against further acute systemic hypotension (15 min). Haemodynamic parameters, arterial blood gases and body temperature were monitored. Energy metabolites were determined in rat parietotemporal cerebral cortex: adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), phosphocreatine (PCr), and adenosine by the high-pressure liquid chromatography (HPLC) technique and lactate spectrophotometrically. After 2 weeks, permanent 2-vo led to a significant decrease in the concentrations of cortical tissue ATP and PCr, from 3.06+/-0.48 to 2. 09+/-0.28 and from 4.27+/-0.63 to 3.35+/-0.41 micromol/g, respectively. These changes were associated with a two-fold increase in AMP and adenosine. Acute systemic hypotension alone (non-preconditioning) reduced ATP and PCr drastically, to 0.97+/-0. 51 and 1.76+/-1.23 micromol/g. Tissue concentrations of lactate, AMP, and adenosine were markedly increased, three- to five-fold, in 'non-preconditioned' brain tissue. In contrast, after 2 weeks of 2-vo acute hypotension did not significantly alter the cortical energy state any further. The effects of preconditioning on tissue ATP and PCr were most pronounced at 5 min and 48 h after reperfusion. In conclusion, permanent 2-vo seems to activate compensatory mechanisms, which effectively protect the rat's cortical energy metabolism against an additional ischaemic attack ('preconditioning-like' effect).     

Interrelation between cerebral energy metabolism and behaviour in a rat model of permanent brain vessel occlusion

The present study investigates the interrelation between cerebral energy metabolism and memory capacities after acute and permanent occlusions of carotid and vertebral arteries in adult Wistar rats (n=60). Tissue ATP, phosphocreatine, ADP, AMP and adenosine concentrations were determined in rat brain by high-pressure liquid chromatography (HPLC) analysis. Lactate and pyruvate were measured spectrophotometrically. Rats underwent psychometric testing by means of a holeboard test, closed field activity, and passive avoidance behaviour. Acute cerebral ischaemia was associated with a substantial deficit in energy load (−50%). Cortical adenosine and lactate exhibited a 7- and a 10-fold increase, respectively, in concentration. After 2 weeks of four-vessel occlusion, cortical ATP and phosphocreatine showed a partial enhancement in their concentrations if compared with acute ischaemia. Consequently, energy load (μmol/g) increased from 0.59 to 1.42 in cerebral cortex and from 0.58 to 1.14 in hippocampus under conditions of acute and permanent ischaemia, respectively. While lactate was normalized, adenosine showed a 2-fold increase in its cortical concentration. All animals improved their abilities in learning, memory and cognition after a 7-day training period. Acute vessel occlusion severely decreased working memory (WM), reference memory (RM) and locomotor activity. Simultaneously, the passive avoidance test showed a significant reduction in latency time from 247±85 s (sham) to 145±132 s. The partial improvement in brain energy state was accompanied by a relative improvement in WM and RM, although both memory capacities remained significantly lower than in controls. The data of the present study demonstrate a linear relationship between cerebral energy metabolism and brain memory capacities after acute and permanent vessel occlusions in rats.     

Deleterious brain cell membrane effects after NMDA receptor antagonist administration to newborn piglets

Previous studies have shown that administration of the N-methyl- -aspartate (NMDA) receptor antagonist 3-(2-carboxypiperazin-4-yl)-1-phosphonic acid (CPP) reduces NMDA-mediated neurotoxicity in animal models of hypoxia/ischemia but also may induce brain tissue vacuolization and alter glucose metabolism. The present study tests the hypothesis that CPP administration alters brain cell membrane structure and function in the cerebral cortex of normoxic newborn piglets through the generation of oxygen free radicals and induction of lipid peroxidation. Twenty six anesthetized, ventilated newborn piglets—13 treated with 2 mg/kg i.v. CPP and 13 untreated controls—were studied. ATP and phosphocreatine (PCr) levels were measured as an index of cellular energy metabolism and tissue glucose levels determined. Na⁺,K⁺-ATPase activity was measured as an index of brain cell membrane function and the lipid peroxidation products conjugated dienes (CD) and fluorescent compounds (FC) measured. Free radical generation was detected on cortical biopsies homogenized with α-phenyl-N-tert-butyl-nitrone (PBN) through electron spin resonance spectroscopy. Signal height of spectrum was divided by dry tissue weight and expressed as mm/g tissue. In the two groups brain tissue ATP and PCr levels were not different. Tissue glucose levels were higher in the CPP group (24±5 mg/dl) than in controls (14±3 mg/dl), p<0.05, whereas Na⁺,K⁺-ATPase activity was lower in the CPP group than in controls (34±4 vs. 43±6 μmol Pi/mg protein/h), p<0.05. Lipid peroxidation products were higher in the CPP group (CD: 57±19 nmol/g brain, FC: 1.5±0.3 μg/g brain) than in controls (CD: 0±0 nmol/g brain, FC: 0.9±0.2 μg/g brain), p<0.05. Free radical intensity was higher in the CPP group (493±397 mm/g tissue) than in controls (51±83 mm/g tissue), p<0.05. In vitro administration of CPP to brain cell membranes did not change Na⁺,K⁺-ATPase activity or the generation of lipid peroxidation products. The data demonstrate that administration of CPP induces lipid peroxidation, results in free radical generation, decreases brain cell membrane Na⁺,K⁺-ATPase activity and alters glucose metabolism in the cerebral cortex of newborn piglets. Since CPP is a potent antagonist of the NMDA receptor, we speculate that CPP generates free radicals through a pathway independent of the NMDA receptor by altering cellular metabolism and possibly glucose utilization during normoxia in newborn piglets.     

Effect of metabotropic glutamate receptor activation on receptor-mediated cyclic AMP responses in primary cultures of rat striatal neurones

Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. -glutamate and the selective group I agonist, (S)-dihydroxyphenylglycine (S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA), with EC₅₀ values of 3.41±0.39 and 5.69±1.64 μM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 μM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, -glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC₅₀ values to those observed for the augmentation of NECA cyclic AMP responses (5.19±1.18 and 3.78±1.42 μM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu₅ receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.     

The protective effect of hypothermia on hippocampal slices from guinea pig during deprivation of oxygen and glucose

Using guinea pig hippocampal slice preparations, the effect of temperature on the electrical activity and the protective effect of hypothermia against deprivation of both oxygen and glucose were studied by recording field potentials of pyramidal cell layer (CA_3–4 area) and by measuring the content of adenosine triphosphate (ATP), phosphocreatine (PCr) and lactate of each slice. Cooling the perfusion medium from 37 to 21 °C caused a decrease in the amplitude of field potentials, although the amplitude increased (120%) transiently at around 33 °C. The electrical activity ceased at around 22 °C. When the temperature was raised from 21 to 37 °C, the activity recovered reversibly. However, when the temperature was raised to above 38 °C, the amplitude decreased and disappeared irreversibly at 42 °C. During deprivation, energy consumption (total P used;2 × ΔATP+ ΔPCr+ 1.3 × Δlactate) was suppressed by lowering the tissue temperature and the initial (0–2.5 min deprivation) energy use rate was calculated to be 42.2 at 37°C, 22.8 at 28 °C and 7.0 at 21 °C (P m mol/kg protein/min), respectively. From these values, Q10 was estimated to be 2.05. With regard to the protective effect of hypothermia, the critical survival time (period of deprivation of oxygen and glucose for the complete recovery in neural activity and the level of high energy phosphates) was 10 min at 37 °C, 15 min at 28 °C, and 45 min at 21 °C, respectively. These results indicate that moderate hypothermia (28 °C) is not very effective in protecting the brain tissue against deprivation of oxygen and glucose, and that deep hypothermia (21 °C) has a much more potent protective effect against deprivation from the functional and metabolic viewpoint.     

Flow thresholds for cerebral energy disturbance and Na+ pump failure as studied by in vivo 31P and 23Na nuclear magnetic resonance spectroscopy

The relationships among CBF, cerebral energy metabolism, Na+ pump activity, and electrocorticograms (ECoG) following graded hypotension were studied in 48 gerbils. Energy metabolism and Na+ pump activity were estimated by in vivo 31P and 23Na nuclear magnetic resonance (NMR) spectroscopy, and CBF was determined by [14C]iodoantipyrine methods at the end of the experiments. The CBF measured in normotensive animals was 0.51 +/- 0.07 ml/g brain/min. Following graded hypotension, no 31P spectral change was observed until CBF fell to 0.21-0.27 ml/g brain/min, at which level the intracellular pH began to decrease in association with ECoG voltage reduction. At a CBF level of 0.18-0.23 ml/g brain/min, phosphocreatine (PCr) began to decrease in association with inorganic phosphate (Pi) elevation. At this level, ECoG became isoelectric, although no adenosine triphosphate (ATP) change yet resulted. At a flow level of 0.12-0.14 ml/g brain/min, ATP began to decrease gradually. At 0.04-0.05 ml/g brain/min, PCr and ATP virtually disappeared, and the 23Na signal intensity suddenly changed. The present study demonstrated flow thresholds for the development of tissue acidosis, PCr-Pi changes, and ATP reduction. It appears that functional suppression occurs prior to ATP changes, whereas Na+ pump failure results after ATP depletion.     

Thyroid hormones in human tumoral and normal nervous tissues

We have studied T4 and T3 concentrations, DNA and protein concentrations and 5′ and 5 deiodinases in samples of brain tumors obtained at surgery from 49 patients, and, in most cases, also from surrounding normal tissue. T4 concentrations in normal cortical tissue (6.19±0.45 ng/g) were lower than in white matter, but the difference disappeared when referred to the DNA content (2.26±0.27 ng/mg DNA). No other differences were found between cortical and white matter, or among cortical lobes. T4 in normal tissue was higher than previously reported, mostly from autopsy samples, whereas T3 (0.99±0.07 ng/g) was similar. 5′D-I activity was negligible as compared to 5′D-II (8.11±1.09 fmol/h/mg protein). When expressed in relation to the different DNA contents of normal vs. tumoral tissue, 5′D-II activities were the same for both. 5D activity was highly variable in the tumoral tissue, with negligible activities in meningiomas and pituitary adenomas. When referred to the DNA content, T4 and 5′D-II were the same, but T3 concentrations were lower in the tumor (0.24±0.03 ng/mg DNA) as compared to normal (0.35±0.04 ng/mg DNA) tissue samples. Whether or not this decrease of T3 affects the expression of T3-sensitive processes remains to be studied.     

Alterations in brain phosphate metabolite concentrations in patients with human immunodeficiency virus infection

Human immunodeficiency virus (HIV)-infected individuals often demonstrate neuropsychiatric impairment; however, it is unclear how brain metabolism may be altered in such patients. We used in vivo phosphorus 31 magnetic resonance spectroscopy to noninvasively assess brain energy and phospholipid metabolism by measuring brain concentrations of adenosine triphosphate (ATP), phosphocreatine (PCr), and inorganic phosphate (Pi), as well as phospholipid compounds and intracellular pH. In study 1, 17 HIV-seropositive men with varying degrees of neuropsychiatric impairment and six control subjects were studied. Localized spectra were obtained from a heterogeneous 5 x 5 x 5-cm volume of interest (VOI). Patients with HIV infection had a significantly lower ATP/Pi ratio and a trend for a lower PCr/Pi ratio than did the control group. In addition, the ATP/Pi and PCr/Pi ratios were both significantly negatively correlated with overall severity of neuropsychiatric impairment. In study 2, three HIV-seropositive men with neuropsychiatric impairment were compared with 11 HIV-seronegative men. Localized phosphorus 31 magnetic resonance spectra were obtained from two relatively homogeneous VOIs: (1) a predominantly white matter VOI, and (2) a predominantly subcortical gray matter VOI. The three HIV-infected patients demonstrated significantly decreased ATP and PCr concentrations in the white matter VOI. These results suggest that HIV infection of the brain may impair brain cellular oxidative metabolism and that the degree of metabolic compromise may be related to the severity of neuropsychiatric impairment.     

Depletion of energy metabolites following acetylcholinesterase inhibitor-induced status epilepticus: protection by antioxidants

Status epilepticus (SE)-induced neuronal injury may involve excitotoxicity, energy impairment and increased generation of reactive oxygen species (ROS). Potential treatment therefore should consider agents that protect mitochondrial function and ROS scavengers. In the present study, we examined whether the spin trapping agent N-tertbutyl-alpha-phenylnitrone (PBN) and the antioxidant vitamin E (DL-alpha-tocopherol) protect levels of high-energy phosphates during SE. In rats, SE was induced by either of two inhibitors of acetylcholinesterase (AChE), the organophosphate diisopropylphosphorofluoridate (DFP, 1.25 mg/kg, sc)- or the carbamate carbofuran (1.25 mg/kg, sc). Rats were sacrificed 1 h or 3 days after onset of seizures by head-focused microwave (power, 10 kW; duration 1.7 s) and levels of the energy-rich phosphates adenosine triphosphate (ATP) and phosphocreatine (PCr) and their metabolites adenosine diphosphate (ADP) and adenosine monophosphate (AMP), and creatine (Cr), respectively, were determined in the cortex, amygdala and hippocampus. Within 1 h of seizure activity, marked declines were seen in ATP (34-60%) and PCr (25-52%). Total adenine nucleotides (TAN = ATP + ADP + AMP) and total creatine compounds (TCC = PCr + Cr) were also reduced (TAN 38-60% and TCC 25-47%). No changes in ATP/AMP ratio were seen. Three days after the onset of seizures, recovery of ATP and PCr was significant in the amygdala and hippocampus, but not in the cortex. Pretreatment of rats with PBN (200 mg/kg, ip, in a single dose), 30 min before DFP or carbofuran administration, prevented induced seizures and partially prevented depletion of high-energy phosphates. Pretreatment with the natural antioxidant vitamin E (100 mg/kg, ip/day for 3 days), partially prevented loss of high energy phosphates without affecting seizures. In controls, citrulline, a product of nitric oxide synthesis, was found to be highest in the amygdala, followed by hippocampus, and lowest in the cortex. DFP- or carbofuran-induced seizures caused elevation of citrulline levels seven- to eight-fold in the cortex and three- to four-fold in the amygdala and hippocampus. These results suggest a close relationship between SE, excitotoxicity and energy metabolism. The involvement of oxidative stress is supported by the findings that DFP and carbofuran trigger an excessive nitric oxide (NO) production in the seizure relevant regions of the brain.     

Delayed treatment with nicotinamide inhibits brain energy depletion, improves cerebral microperfusion, reduces brain infarct volume, but does not alter neurobehavioral outcome following permanent focal cerebral ischemia in Sprague Dawley rats

Delayed treatment with nicotinamide (NAm) reduces infarction induced by middle cerebral artery occlusion (MCAO) in rats. This study explored some potential mechanisms by which delayed NAm treatment may confer protection in the brain of Sprague-Dawley rats following permanent MCAO (pMCAO). NAm (500 mg/kg) or vehicle was given 2 h after the onset of pMCAO. Cortical microperfusion, brain and rectal temperature were serially measured. Neurobehavioral examinations were performed at 24 h post-ischemia followed by sacrifice for histologic assessment. Some rats were also sacrificed at 4 h post-ischemia for analyses of ATP, ADP, AMP, and adenosine. Permanent MCAO induced spontaneous hyperthermia and a sharp decrease in cortical microperfusion, ATP concentration, and the sum of adenine nucleotides (p < 0.05). At 4 h post-ischemia, NAm improved ATP recovery, the sum of adenine nucleotides (p < 0.05) and attenuated the ischemia-induced systemic hyperthermia (p < 0.05) without affecting brain temperature or cortical microperfusion. At 24 h, NAm improved cortical microperfusion in the ischemic hemisphere and reduced total infarct volume (p < 0.05), but did not affect behavioral scores. The data suggest that NAm attenuated brain damage following pMCAo initially by improving cerebral bioenergetic metabolism during the sub-acute phase of ischemia, followed by a delayed improvement in microvascular perfusion.     

Comparison of the somatosensory evoked potential and the direct cortical response following severe incomplete global ischemia: selective vulnerability of the white matter conduction pathways

Eight cats were subjected to graded hemorrhagic hypotension following bilateral carotid ligation to produce incomplete global cerebral ischemia. Three additional cats served as controls. The somatosensory evoked potential (SEP) and direct cortical response (DCR) were monitored in all animals and in each case, the cortical component of the SEP was abolished during progressive ischemia while the morphology of the DCR was well-preserved but with reduced amplitude. Determinations of adenosine triphosphate (ATP), phosphocreatine (PCr), and lactate levels in cerebral cortex and white matter were made in five experimental cats and the three controls. At the time of failure of the cortical SEP, PCr was dramatically reduced and lactate moderately elevated in the white matter while ATP remained unchanged. Cortical lactate was only mildly elevated and PCr and ATP were unchanged accounting for preservation of the DCR. In this model of global ischemia, abolition of the cortical SEP is due to a block of stimulus conduction in white matter projection pathways. A hypothesis to explain the observed metabolic changes is presented and correlation is made to clinical situations.     

Effects of hypoxia on rat brain metabolism: unilateral in vivo carotid infusion

An in vivo brain perfusion technique was used to examine effects of hypoxia on cerebral cortical metabolism in barbiturate-anesthetized rats. Dulbecco's phosphate-buffered solution (PBS), or Dulbecco's PBS + 6 mM glucose, was infused into the right carotid circulation for 0 to 3 min, at a rate that reduced regional cerebral blood flow to the ipsilateral parietal lobe by more than 40% and O2 delivery by about 50%. The duration of infusion of either solution was correlated negatively with the ipsilateral parietal lobe concentrations of glucose, ATP, and phosphocreatine (PCr), and positively with parietal concentrations of lactate and cAMP. cGMP increased in relation to infusion duration of Dulbecco's PBS. Statistically significant elevations of brain lactate occurred after 1 min of infusion of Dulbecco's PBS; lactate was elevated and glucose was reduced after 2 min of infusion of either solution. Brain ATP, PCr, and glycogen concentrations decreased in relation to the elevation in brain lactate, and the [PCr]:[ATP] ratio declined. The results demonstrated that limited hypoxia stimulated cerebral glycolysis and produced a concurrent decrease in brain ATP and PCr. However, ATP was spared to a degree, at the expense of PCr.     

Adenosine stimulates stellation of cultured rat cortical astrocytes

We investigated the effect of adenosine on astrocyte morphology by using cell cultures prepared from the cerebral cortices of neonatal rats. Cultured rat cortical astrocytes exhibited flattened, polygonal morphology in the absence of stimulation, but differentiated into process-bearing stellate cells in response to adenosine (1–1000 μM). Adenosine-induced astrocyte stellation was abolished by treatment with microtubule inhibitors, colchicine and paclitaxel, indicating the involvement of cytoskeletal elements. The effect of adenosine was mimicked by other adenosine receptor agonists, and blocked by adenosine receptor antagonists and guanosine 5′-O-(2-thiodiphosphate), indicating that the effect of adenosine is mediated by G protein-coupled adenosine receptors. Although adenosine receptors are known to be linked to adenylate cyclase or phospholipase C, adenosine did not change intracellular cyclic AMP level nor intracellular Ca²⁺ concentration in astrocytes. Alternatively, adenosine-induced stellation was abolished by tyrosine phosphatase inhibitors, orthovanadate and phenylarsine oxide, suggesting that adenosine causes astrocyte stellation through tyrosine dephosphorylation. Adenosine may function as a factor regulating astrocyte differentiation.     

Maturational changes in intracellular high energy phosphate transport in rat brain.

Maturational changes in intracellular high energy phosphate (HEP) transport in brain were investigated in rats in vivo using 31P nuclear magnetic resonance (NMR) diffusion spectroscopy and saturation transfer experiments. The diffusivities of phosphocreatine (PCr) and adenosine triphosphate (ATP) in adult brain were significantly higher than in newborn pup brain. The estimated diffusion lengths of PCr and ATP were similar in adult and newborn pup brain. The findings indicate firstly that the faster diffusivities of HEP effectively compensates for the higher energy demands in adult brain and secondly that ATP diffusion appears sufficient for brain HEP transport without need for a PCr shuttle mechanism.     

Neuroprotective effects of TNF binding protein in focal cerebral ischemia

The effect of tumor necrosis factor binding protein (TNFbp) was studied in mice subjected to a permanent middle cerebral artery occlusion (MCAO). TNFbp is a dimeric form of the type I soluble TNF receptor linked to polyethylene glycol (TNFbp), and binds and inhibits TNF-α. TNFbp produced a significant reduction in the cortical infarct volume (22.6±3.5 mm³ immediately after MCAO; 25.2±2.4 mm³ 1 h after MCAO) compared with vehicle-treated animals (30.3±3.7 mm³ immediately post MCAO; 31±3.7 mm³ 1 h after MCAO (mean±S.D.) when administered intracranially up to 60 min post-occlusion. The neuroprotective effect of TNFbp was sustained in mice for 2 weeks after MCAO. DNA fragmentation at the margin of the cortical infarcts was dramatically reduced in mice treated with TNFbp whereas all control animals showed consistent and obvious DNA fragmentation 2 weeks after MCAO. TNFbp could have therapeutic value for the treatment of ischemic stroke if the problem of delivery to brain can be overcome.     

In vivo 31phosphorus spectroscopy during transient cerebral ischaemia in the gerbil

The depletion of the high energy phosphates; phosphocreatine and ATP, during cerebral ischaemia disrupts normal cellular function and can lead to cerebral infarction. Using in vivo nuclear magnetic resonance spectroscopy; the metabolic effects of the gerbil model of transient bilateral carotid artery occlusion were quantified. By examining the changes in the inorganic phosphate (Pi), phosphocreatine (PCr) and μ-ATP peaks, the PCr/Pi ratio, the PCr/μ-ATP ratio and intracellular pH (pHi) before, during and after an ischaemic insult were calculated. Preischaemic values for these parameters were: PCr/Pi = 2.466 ± 0.130, PCr/μ-ATP = 1.691 ± 0.053, pHi = 7.112 ± 0.021. By the end of 20 min of global ischaemia, the PCr and μ-ATP peaks fell to levels similar to background in most animals. Calculated values were: PCr/Pi = 0.488 ± 0.126, PCr/μ-ATP = 1.833 ± 0.179, pHi = 6.551 ± 0.258. With reperfusion, PCr/Pi increased rapidly back towards preischaemic levels but pHi improvement was delayed 10 min after that of PCr/Pi. By 1 h of reperfusion, both PCr/Pi and pHi were statistically equivalent to preischaemic values. During ischaemia, ATP was lost more rapidly than the storage form, PCr, but recovery of both was parallel. This suggested an intact ability to store such energy. These data indicate that the gerbil brain recovers normal high energy phosphate levels within an hour following a 20 min ischaemic insult, but that initial reperfusion does not immediately correct intracellular acidosis. Such a delay may prove a useful marker of those animals with more severe ischaemic injury.     

O2 dependence of in vivo brain cytochrome redox responses and energy metabolism in bloodless rats

Oxygen-dependent changes in brain cytochrome redox state and cerebrocortical energy metabolism were evaluated in fluorocarbon-circulated rats at hematocrits of less than 1%. Redox levels of three respiratory chain cytochrome complexes, b, c, and a,a3 (cytochrome c oxidase), were continuously measured directly through the intact skulls of animals using reflectance spectrophotometry. The in vivo redox status of cytochromes at different FiO2 was directly compared with in vitro measured changes in cortical metabolites known to reflect energy production, i.e., glucose, pyruvate, lactate, phosphocreatine (PCr), ADP, and ATP. Lowering the FiO2 to less than 1.0 caused the cytochromes to become increasingly more reduced. This was associated with increased tissue accumulation of pyruvate and lactate and a concomitant increase in the lactate/pyruvate (L/P) ratio. At FiO2 = 0.6, cytochromes b, c, and a,a3 were 57, 53, and 46% reduced, respectively. There was no apparent cerebral energy deficit since changes in cortical PCr, ADP, and ATP concentrations were not statistically significant. Bloodless animals did not survive below FiO2 = 0.5. At this FiO2, the inability of the animals to sustain arterial pressure correlated (r = 0.87) with depletion of PCr and further increases in the L/P ratio (r = 0.66). Yet, the cortical ATP content was reduced by only 9% of control value. These data provide direct evidence that fluorocarbon emulsion (FC-43) sustains brain oxygenation and energy metabolism at high partial pressures of molecular O2. At lower FiO2, however, mitochondrial O2 uptake becomes limited as a function of decreasing perfusion pressure.