Cerebral Phosphoinositide, Triacylglycerol, and Energy Metabolism in Reversible Ischemia: Origin and Fate of Free Fatty Acids

Levels of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), phosphatidic acid, diacylglycerol (DAG), triacylglycerol (TAG), and free fatty acids (FFAs), as well as their fatty acid composition, were determined in rat forebrain during ischemia and postischemic recirculation. Cerebral energy state and electroencephalograms (EEGs) were also studied. Fifteen minutes of ischemia resulted in a decrease in PIP2 and PIP contents but not in PI content, concurrent with an enlargement of the FFA and DAG pools. The latter were enriched in stearate and arachidonate. Prolongation of ischemia did not produce further changes in content of any of the inositol phospholipids, but the increase in levels of FFAs and DAG continued. At the end of 45 min of ischemia, levels of both PIP2 and PIP decreased by 45-50%, and the total phosphoinositide content (PIP2 + PIP + PI) decreased by 21%, whereas levels of FFAs and DAG increased to 14- and 3.6-fold of control levels, respectively. During ischemia, the TAG-palmitate level decreased, but the TAG-arachidonate level increased; the tissue energy state deteriorated severely; and the EEG was suppressed. A 30-min recirculation period after 15 or 45 min of ischemia led to increases in PIP2, PIP, and total phosphoinositide contents, whereas levels of FFAs and DAG promptly decreased toward control values. The TAG-arachidonate level peaked and the TAG-palmitate level returned to a low control value during early recirculation. The ischemic changes in tissue lipids were completely reversed within 3 h of recirculation after both periods of ischemia. Adenylates were fully phosphorylated with as little as 30 min of reflow. The EEG activity partially recovered during reflow after 15 min of ischemia, whereas it remained depressed after prolonged ischemia. Thus, phosphodiesteric cleavage of PIP2 and PIP followed by deacylation of DAG is likely to contribute to the production of FFAs in early ischemia. Deacylation of undetermined lipids plays a role for the increment in levels of FFAs in the later period of ischemia. The rapid postischemic increase in levels of PIP2 and PIP indicates active synthesis not only from existing PI, but probably also by means of accumulated FFAs and DAG. These results indicate that the impaired resynthesis of inositol phospholipids cannot be a cause of the poor EEG activity after prolonged ischemia. Degradation and resynthesis of polyphosphoinositides and formation of TAG-arachidonate may be important for modulation of free arachidonic acid levels in the brain during temporary ischemia.     

Free Fatty Acids and Energy Metabolites in Ischemic Cerebral Cortex with Noradrenaline Depletion

Abstract: We tested whether cerebral noradrenaline (NA) may play a central role in mediating the increased production of free fatty acids (FFAs) during cerebral ischemia. Levels of FFAs, cyclic AMP, and NA, as well as ATP, ADP, and AMP, were measured in cerebral cortex during decapitation ischemia in rats 2 weeks after unilateral locus ceruleus lesion. Comparisons were made between the results obtained from the contralateral cortex with normal NA content and the NA-depleted ipsilateral cortex. Although NA depletion was associated with a diminished transient rise of cyclic AMP in response to ischemia, it failed to influence the magnitude of FFA increase or the decline of energy state within the 15-min period of ischemia. A more than twofold increase of total FFAs (sum of palmitic, stearic, oleic, arachidonic, and docosahexaenoic acids) was observed in both hemispheres at 1 min after decapitation, when energy failure became manifest. The increased production of FFAs continued throughout the 15 min of ischemia, with a preferential rise in the levels of stearic and arachidonic acids. There was an inverse correlation between FFA levels and total adenylate pool. The results do not support a major role for NA and cyclic AMP in increasing cortical FFAs during complete ischemia. Instead, they are consistent with the view that impaired oxidative phosphorylation activates deacylating enzymes. Disturbance of reacylation due to energy depletion is probably another factor contributing to the continuous increase of FFAs during prolonged ischemia.     

Ischemia-Induced Alterations in Lipid Metabolism of the Gerbil Cerebral Cortex: I. Changes in Free Fatty Acid Liberation

Abstract: Does the impaired lipid metabolism during nonlethal transient ischemia truly recover within a few hours after recirculation? In an attempt to answer this question, we first investigated the time course of the changes in the amount and composition of free fatty acids (FFAs) accumulated during 5-min ischemia and after various postischemic recirculation durations (3 min, 1 h, 24 h, 3 days, and 6 days) in the gerbil cerebral cortex. Then those of FFAs liberated in response to the second 5-min ischemia at various recirculation intervals (3 min, 1 h, 3 days, and 6 days) following the initial one were also measured to evaluate the changes in the cellular response. The former study disclosed that the FFA levels transiently returned to the control levels at 1-h recirculation, increased again a few days after the onset of recirculation, followed by the final return to the control levels after 6-day recirculation. The latter study disclosed that the cellular response to the second ischemia was quite different from that to the initial one even after 6-day recirculation, suggesting that membrane lipid metabolism had not yet been recovered even at such a late period. We discuss the significance of the alterations in lipid metabolism.     

Polyphosphoinositides as a Probable Source of Brain Free Fatty Acids Accumulated at the Onset of Ischemia

The quantitative relationship between phosphoinositides and free fatty acids (FFAs) in brain ischemia was studied by measuring contents of individual fatty acids in phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), phosphatidic acid (PA), diacylglycerol (DAG), and the FFA pool. Various periods of complete ischemia (1, 3, 10, and 30 min) were produced by decapitation. Ischemia of 1-3 min caused rapid decreases in PIP2 and PIP content together with preferential production of stearic and arachidonic acids in the DAG and FFA pools. The decrement in levels of these fatty acid residues in polyphosphoinositides was sufficient to account for their increment in levels in the enlarged DAG and FFA pools. After 10 min of ischemia, levels of PIP2, PIP, and DAG approached plateau values, but levels of all FFAs continued to increase. The increases in content of DAG and FFAs at later ischemic periods could not be accounted for by the decreases in content of PIP2 and PIP, PI and PA levels showed only transient and subtle changes. These results indicate that, at the onset of ischemia, phosphodiesteric cleavage of PIP2 and PIP and subsequent deacylation by lipases are primarily responsible for the preferential increase in levels of free stearic and arachidonic acids and that, later, hydrolysis of other phospholipids plays a major role in the continuous accumulation of FFAs.     

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.     

Cyclic AMP Concentrations in Rat Neocortex and Hippocampus During and Following Incomplete Ischemia: Effects of Central Noradrenergic Neurons, Prostaglandins, and Adenosine

The concentrations of cyclic AMP, noradrenaline, glycogen, glucose, lactate, pyruvate, labile phosphate compounds, and free fatty acids were investigated in the rat neocortex and hippocampus during and following cerebral ischemia. An incomplete ischemia of 5 and 15 min duration was induced by bilateral carotid clamping combined with hypotension. The postischemic events were studied after 5, 15, and 60 min of recirculation. Five minutes of ischemia did not significantly alter the neocortical or hippocampal concentrations of cyclic AMP. After 15 min of ischemia the neocortical levels decreased significantly below control values. In the recirculation period following ischemia a significant elevation of the cyclic AMP concentrations was observed. Following 5 min of recirculation after 5 min of ischemia the levels increased from 2.53 +/- 0.21 nmol X g-1 to 5.18 +/- 0.09 nmol X g-1 in the neocortex and from 2.14 +/- 0.16 nmol X g-1 to 3.52 +/- 0.35 nmol X g-1 in the hippocampus. Five minutes of recirculation following 15 min of ischemia led to a significant increase in the levels of cyclic AMP, to 12.86 +/- 1.43 nmol X g-1 in the neocortex to 5.58 +/- 0.57 nmol X g-1 in the hippocampus. With longer recirculation periods the cyclic AMP levels progressively decreased and were similar to control values after 60 min. Depletion of cortical noradrenaline by at least 95% was performed by injections of 6-hydroxydopamine into the ascending axon bundles from the locus ceruleus. The lesion did not significantly change the ischemic or post-ischemic neocortical and hippocampal levels of cyclic AMP, glycogen, or free fatty acids including arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reassessment of Brain Free Fatty Acid Liberation During Global Ischemia and Its Attenuation by Barbiturate Anesthesia

Abstract: We previously reported that whole-brain free fatty acids (FFA) rose almost linearly for up to 1 h after decapitation of unanesthetized rats and was significantly attenuated by pentobarbital anesthesia. However, our values for total FFA and arachidonic, stearic, oleic, and palmitic acids were severalfold higher than those obtained by previous investigators. Based upon the suggestion that this may be due to FFAs released from di- and triglycerides in the quantitation of FFAs, we have now analyzed and improved our procedures for TLC separation of FFA and reassessed the accumulation of FFA in whole brain during decapitation ischemia in unanesthetized and pentobarbital-anesthetized rats. FFA levels in whole brain after 0.5 min of ischemia were one-half to one- fourth the levels previously reported after 1 min of ischemia. The rise in FFA between 0.5 and 60 min of ischemia was 9-fold for total FFA, and between 7 and 12-fold for each of the FFAs quantitated. Pentobarbital significantly attenuated the rise of all FFAs with, however, greater effects on oleic and palmitic acids than previously reported.     

Ischemia-Induced Changes in Cerebral Mitochondrial Free Fatty Acids, Phospholipids, and Respiration in the Rat

Abstract: 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.     

Free Fatty Acids, Neutral Glycerides, and Phosphoglycerides in Transient Focal Cerebral Ischemia

Abstract: Cerebral ischemia is known to cause an increase in levels of free fatty acids (FFAs) and diacylglycerols (DGs), although the mechanism(s) leading to these changes is not well understood. In this study, we examined FFA and DG levels along with those of other lipids in rats during and after transient focal cerebral ischemia induced by temporary occlusion of the right middle cerebral artery (MCA) and both common carotid arteries. During the duration of ischemia (15–60 min), there was a time-dependent increase (two- to 10-fold) in FFA levels in the right MCA cortex, whereas levels of DG and other lipids were not altered appreciably. FFA levels in right MCA cortex returned to near control values after reperfusion. However, following a 60-min ischemic insult, there was a second phase of FFA level increase that was evident after 16 h. The FFAs accumulated during the ischemia period were different from those after reperfusion, suggesting differences in mechanisms for their release. During the second phase of FFA release, there were increases in levels of DGs and triacylglycerols (TGs) with unusually high proportions of 20:4(n-6) and 22:6(n-3). The increases in FFA, DG, and TG levels were marked by a decrease in content of phosphoglycerides (PGs). It is interesting that the increases in levels of FFAs and neutral glycerides accounted only for 10% of the total PGs depleted. The lipid changes during this reperfusion period correlated well with the development of cortical infarct. Because FFAs are potent inhibitors of mitochondrial respiratory function, the time-dependent FFA accumulation during the ischemia period may be an important determinant for the extent of ischemia-induced injury after reperfusion.     

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.     

Effect of the Platelet-Activating Factor Antagonist BN 50739 and Its Diluents on Mitochondrial Respiration and Membrane Lipids During and Following Cerebral Ischemia

Abstract: 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-β-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-β-cyclodextrin was reported to be pharmacologically inactive and capable of dissolving BN 50739. However, hydroxypropyl-β-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 Influence of Bicuculline-Induced Seizures on Free Fatty Acid Concentrations in Cerebral Cortex, Hippocampus, and Cerebellum

Abstract: Using ventilated rats maintained on N₂O-O₂ (70:30, vol/vol) we induced continuous seizures with i.v. bicuculline and analysed free fatty acids (FFA) in cerebral cortex, hippocampus, and cerebellum after seizure durations of 1–120 min. In the cerebral cortex, peak FFA concentrations were observed after 5 min, with a threefold increase in total FFA content. The values then remained unchanged for the next 15-20 min, but decreased thereafter. At 60 and 120 min, total FFA contents were only moderately increased above control. In the initial period, arachidonic acid increased about 10-fold and stearic acid 2- to 3-fold, with little change in palmitic acid and linoleic acid concentrations. At all times, the docosahexenoic acid concentration was markedly increased. Following its massive accumulation at 1 min, arachidonic acid gradually decreased in concentration. Pretreatment of animals with indomethacin did not alter this behaviour. After 20 and 120 min of seizure activity, changes in total and individual FFA concentrations in the hippocampus were similar to those observed in the cerebral cortex. The cerebellum behaved differently. Thus, at 20 min the only significant change was a 5- to 10-fold increase in arachidonic acid concentration and, after 120 min, total and individual FFA concentrations were similar to control values. Furthermore, since the control values for arachidonic acid were much lower in the cerebellum, the 20-min values were only about 20% of those observed in the cerebral cortex and the hippocampus.     

Changes in Cerebral Acyl-CoA Concentrations Following Ischemia-Reperfusion in Awake Gerbils

Abstract: Transient global cerebral ischemia affects phospholipid metabolism and features a considerable increase in unesterified fatty acids. Reincorporation of free fatty acids into membrane phospholipids during reperfusion following transient ischemia depends on conversion of fatty acids to acyl-CoAs via acyl-CoA synthetases and incorporation of the acyl group into lysophospholipids. To study the effect of ischemia-reperfusion on brain fatty acid and acyl-CoA pools, the common carotid arteries were tied for 5 min in awake gerbils, after which the ligatures were released for 5 min and the animals were killed by microwave irradiation. Twenty percent of these animals (two of 10) were excluded from the ischemia-reperfusion group when it was demonstrated statistically that brain unesterified arachidonic acid concentration was not elevated beyond the range of the control group. Brain unesterified fatty acid concentration was increased 4.4-fold in the ischemic-reperfused animals, with stearic acid and arachidonic acid increasing the most among the saturated and polyunsaturated fatty acids, respectively. The total acyl-CoA concentration remained unaffected, indicating that reacylation of membrane lysophospholipids is maintained during recovery. However, there was a substantial increase in the stearoyl- and arachidonoyl-CoA and a marked decrease in palmitoyl- and docosahexaenoyl-CoA. These results suggest that unesterified fatty acid reacylation into phospholipids is reprioritized according to the redistribution in concentration of acyl-CoA molecular species, with incorporation of stearic acid and especially arachidonic acid being favored.     

Chronic administration of ethyl docosahexaenoate decreases mortality and cerebral edema in ischemic gerbils

Dietary docosahexaenoic acid (DHA) intake can decrease the level of membrane arachidonic acid (AA), which is liberated during cerebral ischemia and implicated in the pathogenesis of brain damage. Therefore, in the present study, we investigated the effects of chronic ethyl docosahexaenoate (E-DHA) administration on mortality and cerebral edema induced by transient forebrain ischemia in gerbils. Male Mongolian gerbils were orally pretreated with either E-DHA (100, 150 mg/kg) or vehicle, once a day, for 4 weeks and were subjected to transient forebrain ischemia by bilateral common carotid occlusion for 30 min. The content of brain lipid AA at the termination of treatment, the survival ratio, change of regional cerebral blood flow (rCBF), brain free AA level, thromboxane B(2) (TXB(2)) production and cerebral edema formation following ischemia and reperfusion were evaluated. E-DHA (150 mg/kg) pretreatment significantly increased survival ratio, prevented post-ischemic hypoperfusion and attenuated cerebral edema after reperfusion compared with vehicle, which was well associated with the reduced levels of AA and TXB(2) in the E-DHA treated brain. These data suggest that the effects of E-DHA pretreatment on ischemic mortality and cerebral edema could be due to reduction of free AA liberation and accumulation, and its metabolite synthesis after ischemia and reperfusion by decreasing the content of membrane AA.     

Cerebral Phosphoinositide and Energy Metabolism During and After Insulin-Induced Hypoglycemia

During and after insulin-induced hypoglycemia, changes in levels of cerebral phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA), triacylglycerol (TAG), diacylglycerol (DAG), and free fatty acids (FFAs) as well as the cerebral energy state were studied in relation to the EEG. In hypoglycemic rats with an EEG pattern of quasiperiodic sharp or slow sharp waves, which preceded the development of an isoelectric EEG, PIP2 levels increased significantly, together with a slight decrease in PI content. Levels of the other lipids did not change during this period. The cerebral energy state was affected only slightly in spite of profound decreases in plasma and tissue glucose levels. With 30 min of an isoelectric EEG, levels of all phosphoinositides and PA decreased significantly; total FFA and DAG contents increased seven- and twofold, respectively; the TAG-palmitate level decreased, and that of TAG-arachidonate increased. Plasma and tissue glucose were nearly depleted, and the cerebral energy state deteriorated severely. The increment in fatty acids in the DAG and FFA pools was less than their loss from phosphoinositides and PA, an observation suggesting vascular washout or oxidation of a portion of the FFAs produced. Following 90 min of glucose infusion, PIP and PA levels recovered to control values; however, the PIP2 content exceeded control levels, and that of PI remained below control levels. DAG and FFA contents returned to normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional Studies of Changes in Brain Fatty Acids Following Experimental Ischaemia and Reperfusion in the Gerbil

Regional studies of brain phospholipid metabolism were carried out during a period of ischaemia induced in the gerbil by bilateral carotid occlusion for 60 min. The associated changes in free fatty acids (FFAs) during this period and following recirculation for up to 180 min were noted. Following ischaemia there was a generalised rise in the levels of all FFAs with no selective release of either the unsaturated (arachidonic and docosahexaenoic) or saturated (palmitic and stearic) fatty acids. There were no observed differences between the brain regions studied, which is in contrast to previously reported observations for prostaglandins. There was also no indication of any specific phospholipid fraction being involved in FFA release. This would indicate that the release of FFAs from phospholipids is a nonspecific event, probably due to the action of hydrolytic lipases. Restoration of the circulation resulted in a short, sharp increase (within 5 min) in all FFAs, but in contrast to the observations during ischaemia alone there was a relatively larger rise in the unsaturated FFAs as compared to the saturated FFAs. Following this increase there was a gradual general decline in all FFA levels until 180 min of reperfusion. Since there was no preferential depletion of unsaturated FFAs during reperfusion, when free radical attack is considered to be at its maximum, it is our opinion that free radical peroxidation is unlikely to explain the pathology described in our model.     

Cerebral energy state, mitochondrial function, and redox state measurements in transient ischemia.

Earlier results are reviewed suggesting that transient pronounced, incomplete cerebral ischemia could be more deleterious for the recovery of brain tissue energy state than a complete interruption of the blood flow. Measurements of respiratory function of brain mitochondria, isolated after 30 min of either complete or incomplete ischemia, demonstrated a similar inhibition of respiratory activity and maximal phosphorylation rates in both situations. This inhibition was totally normalized during recirculation after complete ischemia while a further deterioration was found after incomplete ischemia. The in vivo alterations of the cortical tissue distribution of redox states during transient, incomplete ischemia (15--60 min) were measured using a flying spot fluorometer, which gives a real-time and on-line display of the tissue distribution of NADH and oxidized flavoprotein. A reoxidation in both systems was demonstrated during the recirculation period and the distribution of redox states showed no further heterogeneity in the postischemic period as compared to the preischemic distribution. It is concluded that reoxygenation of the brain tissue is possible even after long periods of incomplete ischemia. The normal distribution of redox states during recirculation suggests that mechanisms other than an impaired or inhomogeneous oxygen delivery during the postischemic period are responsible for the failure in recovery of mitochondrial function and tissue energy state.     

Barbiturate Attenuation of Brain Free Fatty Acid Liberation During Global Ischemia

Abstract: To find a biochemical basis for the increased tolerance of the brain to anoxia during barbiturate anesthesia, we studied whole-brain free fatty acids (FFA) at various times after decapitation of awake and pentobarbital-anesthetized rats. Post-decapitation, the brains were kept at 37°C for 1 to 60 min before freezing in liquid N₂. Nonischemic brains were frozen in liquid N₂, using a rapid sampling technique. Whole-brain arachidonic, stearic, oleic, linoleic, and palmitic acids were quantitated by gas-liquid chromatography. In unanesthetized, nonischemic brain, total FFA was 1226 ± 121 nmol/g brain ( n = 12) and was unaffected by pentobarbital anesthesia (1126 ± 86 nmol/g brain, n = 11), except for a reduction in arachidonic acid. Total FFA in unanesthetized and pentobarbital-anesthetized rats transiently declined between 0 and 1 min of ischemia, and then rose linearly for up to 60 min, with consistently lower values in pentobarbital-treated rats, the greatest attenuation being that of arachidonic and stearic acid liberation. Brain FFA liberation during global ischemia is the first known biochemical variable directly correlated with the duration (i.e., severity) of global ischemia. The attenuation of brain FFA liberation and especially of arachidonic and stearic acids may be the biochemical basis of barbiturate attenuation of ischemic brain injury.     

Ischemic Modification of Cerebrocortical Membranes: 5-Hydroxytryptamine Receptors, Fluidity, and Inducible In Vitro Lipid Peroxidation

The effect of ischemia on the properties of 5-hydroxytryptamine1A + B (5-HT1A+B) and 5-hydroxytryptamine1B (5-HT1B) binding sites, physical-state "fluidity" of the membrane, and its susceptibility to peroxidation in vitro was investigated in the cerebral cortex of gerbils. Ischemia was induced by bilateral carotid artery occlusion for 15 min alone or with release for 1 h. Ischemia both with and without reflow decreased the number of 5-HT1A + B and 5-HT1B binding sites, whereas ischemia and reflow altered the affinity for 5-HT1B binding sites. Resistance to the temperature-dependent increase in "fluidity" of the membrane was detected (by fluorescence anisotropy using 1,6-diphenyl-1,3,5-hexatriene as a probe) after ischemia and reflow but not in ischemia alone. Susceptibility of the membranes to Fe2+- and ascorbic acid-stimulated lipid peroxidation in vitro was decreased following ischemia and recirculation only. These findings strongly suggest that the composition and the function of the membrane are markedly disturbed during recirculation after ischemia.     

Brain Cortical Fatty Acids and Phospholipids During and Following Complete and Severe Incomplete Ischemia

Abstract: To explore the possibility that peroxtdative degradation of brain tissue lipid constituents is an important mechanism of irreversible ischemic damage, we measured cortical fatty acids and phospholipids during reversible brain ischemia in the rat. Neither complete nor severe incomplete ischemia (5 and 30 min) caused any measurable breakdown of total or individual fatty acids or phospholipids. Except for a small (and reversible) decrease of inositol plus serine phosphoglycerides in the early postischemic period following 30 min of incomplete ischemia, there were no significant losses of fatty acids or phospholipids during recirculation. Since peroxidation, induced in brain cortical tissue in vitro , characteristically involves degradation of polyenoic fatty acids (arachidonic and docosahexaenoic acids) and of ethanolamine phosphoglycerides, the present in vivo results fail to support the hypothesis that peroxidation of membrane lipids is of primary importance for ischemic brain cell damage. Both complete and severe incomplete ischemia caused a similar increase in the tissue content of free fatty acids (FFA). Thus the FFA pool increased by about 10 times during a 30-min ischemic period, to constitute 1 - 2% of the total fatty acid pool. Since there was a relatively larger increase in polyenoic FFA (especially in arachidonic acid) than in saturated FFA, the release of FFA may be the result of activation of a phospholipase A₂ unbalanced by reesterification. Increased levels of FFA persisted during the initial recirculation period, but a gradual normalization occurred and the ischemic changes were essentially reversed at 30 min after restoration of circulation. The pathophysiological implications of the changes in FFA are discussed with respect to mitochondrial dysfunction, formation of cellular edema and prostaglandin-mediated deterioration of postischemic circulation.