Eva Westerberg

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.

Influence of Acidosis on Lipid Peroxidation in Brain Tissues in vitro

To study the influence of acidosis on free radical formation and lipid peroxidation in brain tissues, homogenates fortified with ferrous ions and, in some experiments, with ascorbic acid were equilibrated with 5–15% O2 at pH values of 7.0, 6.5, 6.0, and 5.0, with subsequent measurements of thiobarbituric acid-reactive (TBAR) material, as well as of water- and lipid-soluble antioxidants (glutathione, ascorbate, and α-tocopherol) and phospholipid-bound fatty acids (FAs). Moderate to marked acidosis (pH 6.5–6.0) was found to grossly exaggerate the formation of TBAR material and the decrease in α-tocopherol content and to enhance degradation of phospholipid-bound, polyenoic FAs. These effects were reversed at pH 5.0, suggesting a pH optimum at pH 6.0–6.5. It is concluded that acidosis of a degree encountered in ischemic brain tissues has the potential of triggering increased free radical formation. This effect may involve increased formation of the protonated form of superoxide radicals, which is strongly prooxidant and lipid soluble, and/or the decompartmentalization of iron bound to cellular macromolecules like ferritin.

Peroxidative Changes in Brain Cortical Fatty Acids and Phospholipids, as Characterized During Fe2+‐ and Ascorbic Acid‐Stimulated Lipid Peroxidation In Vitro

Abstract: The occurrence of peroxidative damage, as distinguished from anaerobic damage, to brain fatty acids and phospholipids was characterized in vitro. Fe2+ and ascorbic acid were used to stimulate peroxidation in cortical homogenates from rat brain incubated with or without oxygen. Lipid peroxidation was established in samples incubated with oxygen by increased diene conjugation, accumulation of thiobarbituric acid‐reactive material (TBAR) and of lipid‐soluble fluorescent products. No peroxidation occurred in samples incubated in the absence of oxygen (100% N2). Lipid peroxidation was characterized by a selective loss of arachidonic acid and docosahexaenoic acid and by degradation of ethanolamine phosphoglyceride, while choline phosphoglyceride did not change. During the course of peroxidation there were parallel increases in products of lipid peroxidation concomitant with the decrease in polyenoic fatty acids. The maximal changes in diene conjugation and TBAR occurred earlier than the maximal changes in fluorescent material and fatty acids. It is concluded that measurements of changes in brain fatty acid and phospholipid composition may be a useful tool to establishment of whether peroxidative damage is important in vivo in situations with a critically reduced oxygen supply. Estimation of lipid‐soluble fluorescence in vivo may also be useful, since it is considered to reflect the accumulation of stable end products of peroxidation.

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.

Lesions of the glutamatergic cortico-striatal projections in the rat ameliorate hypoglycemic brain damage in the striatum

Unilateral ablations of the motor cortex were performed on rats. One to two weeks following the ablation they were subjected to 30 min of reversible insulin-induced hypoglycemic coma. The levels of glutamate, aspartate, gamma-aminobutyric acid (GABA), taurine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and phosphocreatine (PCr) were determined in frozen tissue sections from the superior half of the caudate nucleus. The lesions induced a specific reduction in the levels of glutamate by approx. 10% in the dorsal caudate nucleus ipsilateral to the lesion, while no significant differences in the levels of aspartate, GABA, taurine, ATP, ADP, AMP or PCr were noted. Neuronal necrosis in the caudate nucleus in animals subjected to 30 min of insulin-induced hypoglycemic coma and one week recovery was assessed by light microscopy. Contralateral to the lesion, extensive neuronal necrosis, mainly affecting small and medium-sized neurons, was observed in the dorsal and lateral caudate nucleus. In the caudate ipsilateral to the lesion a complete amelioration of necrosis was noted in areas subjacent to the lesion. The data suggest that hypoglycemic brain damage is induced by excitotoxins such as glutamate or related compounds.

PROTECTION AGAINST ISCHEMIA-INDUCED NEURONAL DAMAGE BY THE ALPHA 2-ADRENOCEPTOR ANTAGONIST IDAZOXAN : INFLUENCE OF TIME OF ADMINISTRATION AND POSSIBLE MECHANISMS OF ACTION

The protective effect of the alpha 2-receptor antagonist idazoxan against neuronal damage in the neocortex and in the hippocampal CA1 region was studied in rats exposed to 10 min of incomplete forebrain ischemia. When administered i.v. immediately after ischemia (0.1 mg/kg) and subsequently for 6 h (10 micrograms/kg/min), idazoxan significantly reduced neuronal damage in the hippocampus (from 84 to 26%) and in the vulnerable parts of the neocortex (from 15 to 1%). The bolus dose alone provided no significant protection. When idazoxan administration was delayed for 30 min, no significant protection was noticed in the neocortex, and the effect in the hippocampus was ambiguous. A transient elevation of plasma corticosterone levels was induced during ischemia. Idazoxan administration for 2 h did not affect postischemic changes in corticosterone levels compared with saline infusion. Idazoxan (10(-7)-10(-4) M) did not influence the in vitro binding to glutamate receptors in brain slices. Thus, the protective effect of idazoxan cannot be explained by suppression of the plasma corticosteroid levels or via an antagonistic effect on glutamate receptors. Idazoxan apparently protects neurons when given during the first hours of postischemic reperfusion, while histopathological necrosis of neurons becomes visible 48-72 h after ischemia. Detrimental processes causing delayed neuronal death occur in the early postischemic phase and can be influenced by adrenoceptor ligands. Idazoxan may protect by several mechanisms but probably exerts its protective postischemic effect mainly through an increased noradrenergic neuronal activity and an elevation of extracellular noradrenaline (NA) levels in the brain. The favorable effects of NA may either be due to inhibition of excitotoxic neurotransmission or activation of survival-promoting and trophic processes.

Regional Differences in Arachidonic Acid Release in Rat Hippocampal CA1 and CA3 Regions during Cerebral Ischemia

Changes in the levels of arachidonic acid during ischemia in selectively vulnerable areas of the hippocampus were studied in the rat brain. Since neurons in the CA, region are more vulnerable to ischemia than neurons in the adjacent CA3 region, the release of arachidonic acid in these two regions was measured during decapitation ischemia of 4- to 12-min duration. The concentration of free arachidonic acid increased with the duration of ischemia in both regions. However, the level was significantly higher in CA1, than in CA3 after 8 and 12 min of ischemia. This difference in arachidonic acid accumulation may reflect differences between the regions in agonist-dependent phospholipid breakdown as well as calcium-dependent phospholipase activity. The importance for the development of neuronal necrosis is discussed.

Excitatory amino acid receptors and ischemic brain damage in the rat

The excitatory amino acid glutamate has been suggested to be an important mediator of the selective CA1 hippocampal damage which follows transient cerebral ischemia. In order to evaluate the possible involvement of altered glutamate receptor regulation in the expression of the delayed neuronal necrosis following ischemia, we have determined the density of glutamate receptor subtypes in the rat hippocampus following transient ischemia. We report a transient reversible decrease in [3H]AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) binding sites (presumably representing quisqualate receptors) followed by a long term loss of binding at 2 days postischemia which precedes neuronal loss. In contrast, no change was noted in the N-methyl-D-aspartate or kainic acid binding sites over this time period.

Effect of insulin-induced hypoglycemia on the concentrations of glutamate and related amino acids and energy metabolites in the intact and decorticated rat neostriatum

Abstract The glutamate (Glu) terminals in rat neostriatum were removed by a unilateral frontal decortication. One to two weeks later the effects of insulin‐induced hypoglycemia on the steady‐state levels of amino acids [Glu, glutamine (Gin), aspartate (Asp), γ‐aminobutyric acid (GABA), tau‐rine] and energy metabolites (glucose, glycogen, α‐ketoglu‐tarate, pyruvate, lactate, ATP, ADP, AMP, phosphocre‐atine) were examined in the intact and decorticated neostriatum from brains frozen in situ. The changes in the metabolite levels were examined during normoglycemia, hypoglycemia with burst‐suppression (BS) EEG, after 5 and 30 min of hypoglycemic coma with isoelectric EEG, and 1 h of recovery following 30 min of isoelectric EEG. In normoglycemia Glu decreased and Gin and glycogen increased significantly on the decorticated side. During the BS period no significant differences in the measured compounds were noted between the two sides. After 5 min of isoelectric EEG Glu, Gin, GABA, and ATP levels were significantly lower and Asp higher on the intact than on the decorticated side. No differences between the two sides were found after 30 min of isoelectric EEG. After 1 h of recovery from 30 min of isoelectric EEG Glu, Gin, and glycogen had not reached their control levels. Glu was significantly lower, and Gin and glycogen higher on the decorticated side. The Asp and GABA levels were not significantly different from control levels. The results indicate that the turnover of Glu is higher in the intact than in decorticated neostriatum during profound hypoglycemia.

Changes in Regional Neurotransmitter Amino Acid Levels in Rat Brain During Seizures Induced by l‐Allylglycine, Bicuculline, and Kainic Acid

Abstract: Changes in amino acid concentrations were studied in the cortex, cerebellum, and hippocampus of the rat brain, after 20 min of seizure activity induced by kainic acid, 47 μmol/kg i.v.;l‐allylglycine, 2.4 mmol/kg i.v.; or bicuculline, 3.27 μmol/kg i.v. in paralysed, mechanically ventilated animals. Metabolic changes associated with kainic acid seizures predominate in the hippocampus, where there are decreases in aspartate (‐26%), glutamate (‐ 45%), taurine (‐20%), and glutamine (‐32%) concentrations and an increase in ‐γ‐aminobutyric acid (GABA) concentration (+26%). l‐Allylglycine seizures are associated with generalized decreases in GABA concentrations (‐32 to ‐54%), increases in glutamine concentrations (+ 10 to +53%), and a decrease in cortical aspartate concentration (‐14%). Bicuculline seizures, in fasted rats, are associated with marked increases in the levels of hippocampal GABA (+106%) and taurine (+ 40%). In the cerebellum, there are increases in glutamine (+ 50%) and taurine concentrations (+ 36%). These changes can be explained partially in terms of known biochemical and neurophysiological mechanisms, but uncertainties remain, particularly concerning the cer‐ ebellar changes and the effects of kainic acid on dicar‐ boxylic amino acid metabolism.