A novel neuroprotective compound FR901459 with dual inhibition of calcineurin and cyclophilins.

Brain ischemia leads to severe damage in the form of delayed neuronal cell death. In our study, we show that the marked neuroprotection of the new immunosuppressant FR901495 in forebrain ischemia is due not only to inhibition of calcineurin, but also to protection against mitochondrial damage caused by mitochondrial permeability transition pore formation through cyclophilin D, one of the prolyl cis/trans isomerase family members. These findings shed light on the clinical application and development of new drugs for the treatment of ischemic damage in the brain as well as in the heart and liver.

Pathogenesis of hippocampal neuronal death after hypoxia-ischemia changes during brain development.

Transient hypoxia-ischemia (HI) leads to delayed neuronal death in both mature and immature neurons but the underlying mechanisms are not fully understood. To understand whether the pathogenesis of HI-induced neuronal death is different between mature and immature neurons, we used a rat HI model at postnatal days 7 (P7), 15 (P15), 26 (P26) and 60 (P60) in order to investigate ultrastructural changes and active caspase-3 distribution in HI-injured neurons as a function of developmental age. In P7 pups, despite more than 95% of HI-injured neurons highly expressing active caspase-3, most of these active caspase-3-positive neurons revealed mixed features of apoptosis and necrosis (a chimera type) under electron microscopy (EM). Classical apoptosis was observed only in small populations of HI-injured P7 neurons. Furthermore, in rats older than P7, most HI-injured neurons displayed features of necrotic cell death under EM and, concomitantly, active caspase-3-positive neurons after HI declined dramatically. Classical apoptosis after HI was rarely found in neurons older than P15. In P60 rats, virtually all HI-injured neurons showed the shrinkage necrotic morphology under EM and were negative for active caspase-3. These results strongly suggest that pathogenesis of HI-induced neuronal death is shifting from apoptosis to necrosis during brain development.

NXY-059, a nitrone with free radical trapping properties inhibits release of cytochrome c after focal cerebral ischemia.

Recent studies have demonstrated that disodium 2,4-disulfophenyl-N-tert-butylnitrone (NXY-059), a novel nitrone with free radical trapping properties, has a considerable neuroprotective effect against cerebral ischemic injury. The mechanisms of its action have not been fully defined. In order to evaluate whether NXY-059 exerts its protective effects by inhibiting the release of cytochrome c, a key initiator of programmed cell death pathway, we have studied the effects of NXY-059 on reducing infarct volume and on inhibiting cytochrome c release from the mitochondria after transient focal cerebral ischemia. Wistar rats were subjected to 2 hr of middle cerebral artery occlusion and perfusion-fixed after 4, 6, 12, and 24 hr of reperfusion. NXY-059 (30 mg/kg) was i.v. injected 1 hr after reperfusion and followed immediately by 30 mg/kg/hr continuous i.v. infusion for the entire reperfusion period. The results showed that NXY-059 reduced infarct volume from 37.2% to 12.5% (p<0.0001). Immunocytochemistry demonstrated that the release of cytochrome c increased at 6 hr, peaked at 12 and 24 hr of reperfusion. NXY-059 treatment prevented ischemia-induced cytochrome c release. NXY-059 may reduce ischemic brain damage through suppressing the cell death pathway that is initiated by cytochrome c release.

Acidosis promotes the permeability transition in energized mitochondria: implications for reperfusion injury.

We have studied the influence of pH on opening of the mitochondrial permeability transition pore (PTP) in both deenergized and energized mitochondria in the presence of Pi. In deenergized mitochondria from rat brain and heart, we observed the expected inhibition of Ca2+-induced PTP opening at increasingly acidic pH values. Unexpectedly, mitochondria energized with either electron transport complex I or complex II substrates displayed the opposite behavior, acidic pH promoting rather than inhibiting PTP opening. We show that the potentiating effect of acidic pH is due to an increased rate of Pi uptake. The data also revealed that brain mitochondria are more heterogeneous than heart or liver mitochondria in relation to onset of a permeability transition, and that this heterogeneity depends on their Pi transport capacity. Taken together, these results indicate that the inhibitory effects of acidic pH on the PTP may be overcome in situ by an increased rate of Pi uptake, and that ischemic and postischemic acidosis may worsen rather than relieve PTP-dependent tissue damage.

Does long-term glucose infusion reduce brain damage after transient cerebral ischemia?

A recent study reported that hyperglycemia of a brief duration worsens, and of long duration reduces, ischemic brain damage. To test whether this is a valid conception, we induced 10 min of transient forebrain ischemia, recorded postischemic seizures, and evaluated brain morphology. The results showed that administration of glucose 2 h before ischemia aggravated brain damage, induced seizures, and caused animal death in the same manner as was previously observed when glucose was given 30 min before ischemia. Thus, the conclusion that the influence of glucose on an ischemic transient is dependent upon the duration of hyperglycemia is unsubstantiated.

Protein aggregation after focal brain ischemia and reperfusion.

Two hours of transient focal brain ischemia causes acute neuronal death in the striatal core region and a somewhat more delayed type of neuronal death in neocortex. The objective of the current study was to investigate protein aggregation and neuronal death after focal brain ischemia in rats. Brain ischemia was induced by 2 hours of middle cerebral artery occlusion. Protein aggregation was analyzed by electron microscopy, laser-scanning confocal microscopy, and Western blotting. Two hours of focal brain ischemia induced protein aggregation in ischemic neocortical neurons at 1 hour of reperfusion, and protein aggregation persisted until neuronal death at 24 hours of reperfusion. Protein aggregates were found in the neuronal soma, dendrites, and axons, and they were associated with intracellular membranous structures during the postischemic phase. High-resolution confocal microscopy showed that clumped protein aggregates surrounding nuclei and along dendrites were formed after brain ischemia. On Western blots, ubiquitinated proteins (ubi-proteins) were dramatically increased in neocortical tissues in the postischemic phase. The ubi-proteins were Triton-insoluble, indicating that they might be irreversibly aggregated. The formation of ubi-protein aggregates after ischemia correlated well with the observed decrease in free ubiquitin and neuronal death. The authors concluded that proteins are severely damaged and aggregated in neurons after focal ischemia. The authors propose that protein damage or aggregation may contribute to ischemic neuronal death.

Hyperglycemia enhances DNA fragmentation after transient cerebral ischemia.

Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3'-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.

Cyclosporin A, but not FK506, prevents the downregulation of phosphorylated Akt after transient focal ischemia in the rat.

The two immunosuppressants, cyclosporin A (CsA) and FK506, when given 1 and 3 h after the start of reperfusion following 2 h of middle cerebral artery (MCA) occlusion, reduce infarct volume to 30% of control. This suggests a common effect, e.g. one due to suppression of the activation of calcineurin. However, when given by the intracarotid (i.c.) route after only 5 min of recirculation CsA, but not FK506, reduced infarct volume even further, to 10% of control. This was attributed to the fact that CsA, but not FK506, block the in vitro assembly of a mitochondrial permeability transition (MPT) pore. The present experiments were undertaken to further characterize the anti-ischemic effect of CsA, when given i.c. 5 min after recirculation and to explore why CsA, when given at that time, is more efficacious than FK506. It was established that the i.c. administration of CsA in a dose of 10 mg/kg increased the tissue concentration of CsA 2- to 3-fold, when compared to the i.v. administration. CsA proved to be effective in reducing infarct volume even when the tissue damage was assessed by histopathology after 7 days of recovery. MCA occlusion of 2 h duration caused a sustained decrease in the phosphorylation Akt at threonine 308. Since this down regulation of Akt was prevented by CsA, the results suggested a link between dephosphorylaltion of Bad, and cell death. Interestingly FK506 did not prevent down regulation of Akt, it thus seems unlikely that the anti-ischemic effect of CsA is related to its association with cytosolic cyclophilin.

Early release of cytochrome C and activation of caspase-3 in hyperglycemic rats subjected to transient forebrain ischemia.

The mechanisms underlying the aggravating effect of hyperglycemia on brain damage are still elusive. The present study was designed to test our hypothesis that hyperglycemia-mediated damage is caused by mitochondrial dysfunction with mitochondrial release of cytochrome c (cyt c) to the cytoplasm, which leads to activation of caspase-3, the executioner of cell death. We induced 15 min of forebrain ischemia, followed by 0.5, 1, and 3 h of recirculation in sham, normoglycemic and hyperglycemic rats. Release of cyt c was observed in the neocortex and CA3 in hyperglycemic rats after only 0.5 h of reperfusion, when no obvious neuronal damage was observed. The release of cyt c persisted after 1 and 3 h of reperfusion. Activation of caspase-3 was observed after 1 and 3 h of recovery in hyperglycemic animals. No cyt c release or caspase-3 activation was observed in sham-operated controls while a mild increase of cyt c was observed in normoglycemic ischemic animals after 1 and 3 h of reperfusion. The findings that there is caspase activation and cyt c relocation support a notion that the biochemical changes that constitute programmed cell death occur after ischemia and contribute, at least in part, to hyperglycemia-aggravated ischemic neuronal death.

Effects of intracarotid arterial injection of cyclosporin A and spontaneous hypothermia on brain damage incurred after a long period of global ischemia.

A recent study showed that a single intracarotid arterial injection of cyclosporin A (CsA) can dramatically reduce infarct volume in rats subjected to transient focal ischemia. The present experiments were undertaken to investigate whether intracarotid arterial injection of CsA reduces brain damage after global ischemia. Since hypothermia is also an efficacious factor in preventing ischemic brain damage, in the second part of the experiments we tested whether a combination of hypothermia and CsA would provide additional brain protection. Global ischemia of a 30-min duration was induced in the rat. CsA (10 mg/kg) was injected into the carotid artery immediately after reperfusion. Hypothermia was instituted after ischemia by allowing spontaneous head temperature to fall to 30-32 degrees C, while body temperature was upheld at 37 degrees C. The results demonstrated that vehicle-treated animals could not survive beyond 1-2 days after reperfusion, and the histopathological outcome in a separate group of rats perfusion-fixed after 1 day reperfusion showed 80-100% brain damage in the caudoputamen, and in the hippocampal CA1, CA3, CA4 and dentate gyrus subregions. Microinfarction and grade 3 damage were frequently observed in the cingulate and parietal cortex and in the thalamus. CsA moderately prolonged animal survival to 3 days after reperfusion and reduced brain damage to grade 2 in the cortical areas and the thalamus. Hypothermia further increased animal survival to at least 6 days after reperfusion and reduced brain damage to 30% in the caudoputamen, to close to zero in the CA3, CA4, and dentate gyrus, and to grade 1-2 in the cortical areas and the thalamus. The combination of hypothermia and CsA did not give additional protection.

Phosphorylation of extracellular signal-regulated kinase after transient cerebral ischemia in hyperglycemic rats.

The present study was undertaken to investigate whether extracellular signal-regulated kinase (ERK) was involved in mediating hyperglycemia-exaggerated cerebral ischemic damage. Phosphorylation of ERK 1/2 was studied by immunocytochemistry and by Western blot analyses. Rats were subjected to 15 min of forebrain ischemia, followed by 0.5, 1, and 3 h of reperfusion under normoglycemic and hyperglycemic conditions. The results showed that in normoglycemic animals, moderate phosphorylation of ERK 1/2 was transiently induced after 0.5 h of recovery in cingulate cortex and in dentate gyrus, returning to control values thereafter. In hyperglycemic animals, phosphorylation of ERK 1/2 was markedly increased in the cingulate cortex and dentate gyrus after 0.5 h of recovery, the increases being sustained for at least 3 h after reperfusion. Hyperglycemia also induced phosphorylation of ERK 1/2 in the hippocampal CA3 sector but not in the CA1 area. Thus, the distribution of phospho-ERK 1/2 coincides with hyperglycemia-recruited damage structures. The results suggest that hyperglycemia may influence the outcome of an ischemic insult by modulating signal transduction pathways involving ERK 1/2.

Alterations of Akt1 (PKBalpha) and p70(S6K) in transient focal ischemia.

The serine-threonine kinase Akt1 promotes cell survival through inhibition of apoptosis. One of the potential downstream targets of Akt1 is p70 S6 kinase, p70(S6K), an enzyme implicated in the regulation of protein synthesis. In this study, we investigated the changes in total and phosphorylated levels of Akt1 and p70(S6K) during transient focal ischemia. Male Wistar rats were subjected to 2 h of middle cerebral artery occlusion followed by 1, 4, and 24 h of reperfusion. The expression of total and phosphorylated forms of Akt1 and p70(S6K) were examined by Western blot analysis. Phosphorylation of Akt1 on Ser473 transiently increased at 1 and 4 h of reperfusion, whereas phosphorylation of Akt1 on Thr308 was reduced during reperfusion. The levels of total Akt1 remained unchanged at 1 and 4 h of reperfusion, but decreased significantly at 24 h of reperfusion. Phosphorylation of p70(S6K) on Thr389 decreased at 1, 4, and 24 h of reperfusion, while the levels of total p70(S6K) protein remained unchanged at 1 and 4 h of reperfusion but decreased at 24 h of reperfusion. The results show that cell survival pathways, such as Akt1 and p70(S6K) signaling, are suppressed after transient focal ischemia, which may contribute to the development of neuronal cell death after an ischemic insult.

Alteration of cyclic adenosine monophosphate response element binding protein in rat brain after hypoglycemic coma.

In the current study, the temporal and regional changes of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB) were investigated in rat brains subjected to 30 minutes of hypoglycemic coma followed by varied periods of recovery using Western blot and confocal microscopy. The total amount of CREB was not altered in any area examined after coma. The level of the phosphorylated form of CREB decreased during coma but rebounded after recovery. In the relatively resistant areas, such as the inner layers of the neocortex and the inner and outer blades of the dentate gyms (DG), phospho-CREB increased greater than the control level after 30 minutes of recovery and continued to increase up to 3 hours of recovery. In contrast, little or no increase of phospho-CREB was observed during the recovery period in the outer layers of the neocortex and at the tip of the DG, that is, regions that are selectively vulnerable to hypoglycemic insults. The current findings suggest that a neuroprotective signaling pathway may be more activated in the resistant regions than in the vulnerable ones after hypoglycemic coma.

Alterations in lipid and calcium metabolism associated with seizure activity in the postischemic brain.

Transient ischemia is known to lead to a long-lasting depression of cerebral metabolic rate and blood flow and to an attenuated metabolic and circulatory response to physiological stimuli. However, the corresponding responses to induced seizures are retained, demonstrating preserved metabolic and circulatory capacity. The objective of the present study was to explore how a preceding period of ischemia (15 min) alters the release of free fatty acids (FFAs) and diacylglycerides (DAGs), the formation of cyclic nucleotides, and the influx/efflux of Ca(2+), following intense neuronal stimulation. For that purpose, seizure activity was induced with bicuculline for 30 s or 5 min at 6 h after the ischemia. Extracellular Ca(2+) concentration (Ca(2+)(e)) was recorded, and the tissue was frozen in situ for measurements of levels of FFAs, DAGs, and cyclic nucleotides. Six hours after ischemia, the FFA concentrations were normalized, but there was a lowering of the content of 20:4 in the DAG fraction. Cyclic AMP levels returned to normal values, but cyclic GMP content was reduced. Seizures induced in postischemic animals showed similar changes in Ca(2+)(e), as well as in levels of FFAs, DAGs, and cyclic nucleotides, as did seizures induced in nonischemic control animals, with the exception of an attenuated rise in 20:4 content in the DAG fraction. We conclude that, at least in the neocortex, seizure-induced phospholipid hydrolysis and cyclic cAMP/cyclic GMP formation are not altered by a preceding period of ischemia, nor is there a change in the influx/efflux of Ca(2+) during seizure discharge or in associated spreading depression.

Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation.

The involvement of caspase-3 in cell death after hypoxia-ischemia (HI) was studied during brain maturation. Unilateral HI was produced in rats at postnatal day 7 (P7), 15 (P15), 26 (P26), and 60 (P60) by a combination of left carotid artery ligation and systemic hypoxia (8% O2). Activation of caspase-3 and cell death was examined in situ by high-resolution confocal microscopy with anti-active caspase-3 antibody and propidium iodide and by biochemical analysis. The active caspase-3 positive neurons were composed of more than 90% HI damaged striatal and neocortical neurons in P7 pups, but that number was reduced to approximately 65% in striatum and 34% in the neocortex of P15 pups, and approximately 26% in striatum and 2% in neocortex of P26 rats. In P60 rats, less than 4% of the damaged neurons in striatum and less than 1% in neocortex were positive for active caspase-3. Western blot analysis demonstrated that the level of inactive caspase-3 in normal forebrain tissue gradually declined from a high level in young pups to very low levels in adult rats. Concomitantly, HI-induced active caspase-3 was reduced from a relatively high level in P7, to moderate levels in P15 and P26, to a barely detectable level in P60 rats. The authors conclude that the involvement of caspase-3 in the pathogenesis of cell death after HI declines during neuronal maturation. The authors hypothesize that caspase-3 may play a major role in cell death in immature neurons but a minor role in cell death in mature neurons after brain injury.

Differential phosphorylation at Ser473 and Thr308 of Akt-1 in rat brain following hypoglycemic coma.

We analyzed both total Akt-1 and phosphorylation of Akt-1 at residues Ser473 and Thr308 (phospho-Akt-1(Ser474) and phospho-Akt-1(Thr308), respectively) in the outer and inner layers of cortex following 30 min of hypoglycemic coma by Western blot analyses and confocal microscopy. The total amount of Akt-1 was not altered in any area examined. Phospho-Akt-1(Ser474), however, increased significantly in both layers of cortex at 0 and 30 min of recovery, but returned to control level at 3 h of recovery. In the vulnerable area (outer layer of cortex), no upregulation of phospho-Akt-1(Thr308) was observed at any time points examined. In the resistant area like inner layer of cortex, however, phospho-Akt-1(Thr308) was significantly over the control level at 3 h of recovery. Confocal microscopy result indicates that most of phospho-Akt-1(Thr308) had already moved into nucleus at 3 h of recovery. Our results suggest that Akt-1, when phosphorylated at Thr308, may play a protective role for neurons in the resistant regions of the brain.

Cyclosporin A enhances survival, ameliorates brain damage, and prevents secondary mitochondrial dysfunction after a 30-minute period of transient cerebral ischemia.

Cyclosporin A (CsA) has been shown to be efficacious in protecting against ischemic injury after short periods (5 to 10 min) of forebrain ischemia. The present experiments were undertaken to study if a long period of forebrain ischemia (30 min), induced at a brain temperature of 37 degrees C, is compatible with survival and if the brain damage incurred can be ameliorated by CsA. The results showed that animals subjected to 30 min of forebrain ischemia at a brain temperature of 37 degrees C failed to survive after the first 24 h of recovery and showed extensive neuronal necrosis in all selectively vulnerable regions after 1 day of survival. CsA, when injected in combination with an intracerebral lesion to open the blood-brain barrier, markedly prolonged the survival time. CsA-injected animals also showed amelioration of histological lesions, an effect that was sustained for at least 4 days. Experiments with mitochondria isolated from the neocortex and hippocampus showed that state 3 respiratory rates decreased during ischemia, recovered after 1 and 3 h of recirculation, and then showed a secondary decline at 6 h. Administration of CsA prevented this secondary decline. Measurements of neocortical cerebral blood flow showed that there was no secondary hypoperfusion prior to secondary mitochondrial dysfunction, implying that changes in blood flow may not be responsible for the rapidly developing, secondary brain damage. The results thus demonstrate that if brain temperature is upheld at 37 degrees C, a 30-min period of ischemia is not compatible with survival after the first day of recovery, and gross histopathological damage develops within that period. CsA was efficacious in prolonging animal survival, ameliorating brain damage, and preventing the secondary mitochondrial dysfunction. Since CsA blocks the mitochondrial permeability transition pore its action may, at least in part, be on mitochondrial integrity and function.

Is neuronal injury caused by hypoglycemic coma of the necrotic or apoptotic type?

In this study, we explored if a 30 minute period of hypoglycemic coma yields damage which shows some features associated with apoptosis. To that end, we induced insulin-hypoglycemic coma of 30 min duration, and studied brain tissues after the coma period, and after recovery period of 30 min, 3 h, and 6 h. Histopathological data confirmed neuronal damage in all of the vulnerable neuronal populations. Release of cytochrome c (cyt c), assessed by Western Blot, was observed in the neocortex and caudoputamen after 3 and 6 h of recovery. In these regions, the caspase-like activity increased above control after 6 h of recovery. By laser-scanning confocal microscopy, a clear expression of Bax was observed after 30 min of coma in the superficial layers of the neocortex, reaching a peak after 30 min of recovery. Punctuate immunolabeling surrounding nuclei in soma and dendrites in cortical pyramidal neurons likely represents mitochondria, which suggests that Bax protein assembled at the surface of mitochondria in vulnerable neocortical neurons. It is concluded that although previous morphological data have suggested that cells die by necrosis, neuronal damage after hypoglycemic coma shows some features of apoptosis.

Characteristics of the calcium-triggered mitochondrial permeability transition in nonsynaptic brain mitochondria: effect of cyclosporin A and ubiquinone O.

The objective of the present study was to assess the capacity of nonsynaptic brain mitochondria to accumulate Ca2+ when subjected to repeated Ca2+ loads, and to explore under what conditions a mitochondrial permeability transition (MPT) pore is assembled. The effects of cyclosporin A (CsA) on Ca2+ accumulation and MPT pore assembly were compared with those obtained with ubiquinone 0 (Ubo), a quinone that is a stronger MPT blocker than CsA, when tested on muscle and liver mitochondria. When suspended in a solution containing phosphate (2 mM) and Mg2+ (1 mM), but no ATP or ADP, the brain mitochondria had a limited capacity to accumulate Ca2+ (210 nmol/mg of mitochondrial protein). Furthermore, when repeated Ca2+ pulses (40 nmol/mg of protein each) saturated the uptake system, the mitochondria failed to release the Ca2+ accumulated. However, in each instance, the first Ca2+ pulse was accompanied by a moderate release of Ca2+, a release that was not observed during the subsequent pulses. The initial release was accompanied by a relatively marked depolarization, and by swelling, as assessed by light-scattering measurements. However, as the swelling was <50% of that observed following addition of alamethicin, it is concluded that the first Ca2+ pulse gives rise to an MPT in a subfraction of the mitochondrial population. CsA, an avid blocker of the MPT pore, only marginally increased the Ca(2+)-sequestrating capacity of the mitochondria. However, CsA eliminated the Ca2+ release accompanying the first Ca2+ pulse. The effects of CsA were shared by Ubo, but when the concentration of Ubo exceeded 20 microM, it proved toxic. The results thus suggest that brain mitochondria are different from those derived from a variety of other sources. The major difference is that a fraction of the brain mitochondria, studied presently, depolarized and showed signs of an MPT. This fraction, but not the remaining ones, contributed to the chemically and electron microscopically verified mitochondrial swelling.

Effect of alpha-phenyl-N-tert-butyl nitrone (PBN) on fetal cerebral energy metabolism during intrauterine ischemia and reperfusion in rats.

The objective of the present study was to explore whether a free radical spin trap agent, alpha-phenyl-N-tert-butyl nitrone (PBN), influences bioenergetic failure induced in the 20-day-old fetal brain by 30 min of intrauterine ischemia in Wistar rats. Fetal brains were frozen in situ at the end of ischemia and after 1, 2, and 4 h of recirculation for analysis of ATP, ADP, AMP, and lactate. PBN or vehicle was given 1 h after recirculation. Tissue oxygen tension was evaluated in placental and fetal cerebral tissues throughout the whole periods of 30 min of ischemia and 4 h of recirculation. Ischemia was associated with a decrease in ATP concentration and an increase in lactate concentration (p < 0.001). Recirculation (1 and 2 h) led to a recovery of ATP concentration, but continued reflow (4 h) was associated with a secondary deterioration of high-energy phosphates (p < 0.01). Lactate concentration increased during this recovery period. This deterioration was prevented by PBN (p < 0.05). After 30 min of ischemia, tissue oxygen tension in placenta and fetal brain decreased to about 30% and 50% of control, respectively. However, recirculation brought about a recovery of oxygen delivery. The results indicate that although during the early time period after ischemia fetal cerebral energy metabolism is maintained by an acceleration of the anaerobic glycolytic rate, secondary deterioration of cellular bioenergetic state develops in the immature fetal brain. This deterioration may be due to mitochondrial dysfunction, which may be induced by oxygen-derived free radicals, and not by compromised microcirculation.