Jaroslava Folbergrová

Focal and Perifocal Changes in Tissue Energy State during Middle Cerebral Artery Occlusion in Normo- and Hyperglycemic Rats

The objective of the present study was to assess changes in cellular energy metabolism in focal and perifocal areas of a stroke lesion and to explore how these changes are modulated by preischemic hyperglycemia. A model for reversible occlusion of the middle cerebral artery (MCA) in rats was used to study changes in energy metabolism. Following MCA occlusion for 5, 15, or 30 min in normoglycemic rats, the tissue was frozen in situ, and samples from the lateral caudoputamen and from two neocortical areas were collected for metabolite analyses, together with a control sample from the contralateral, nonischemic hemisphere. Two other groups, subjected to 30 min of MCA occlusion, were made hyperglycemic by acute glucose infusion or by prior injection of streptozotocin. Enzymatic techniques were used for measurements of phosphocreatine, creatine, ATP, ADP, AMP, glycogen, glucose, pyruvate, and lactate. The neocortex of the contralateral, nonischemic hemisphere had labile metabolites that were similar to those measured in control animals. Ipsilateral neocortex bordering the focus, and thus constituting the “penumbra,” showed mild to moderate ischemic changes. In the “focus” (lateral caudoputamen plus the overlying neocortex), deterioration of energy state was rapid and relatively extensive (ATP content 20–40% of control). After 5 min of occlusion, no further deterioration of metabolic parameters was observed. Substrate levels were markedly reduced, and lactate content rose to ∼10 mM kg−1. In the animals with the most severe energy depletion, no additional accumulation of lactate occurred, suggesting substrate depletion. This was confirmed by the results obtained in the hyperglycemic subjects whose tissue lactate contents rose to ∼20 mM kg−1. However, the energy state of the focus was better preserved in both hyperglycemic groups as compared with the normoglycemic group. It has been shown, in this model, that relatively brief occlusion periods are required to induce infarction. The present results demonstrate that this can occur in spite of the absence of pronounced depletion of energy reserves. After 30 min of MCA occlusion, infarction developed in the lateral caudoputamen, but not in the neocortex. Since a similar perturbation in metabolic state was demonstrated here, other factors must contribute to the degree of tissue damage. The present results suggest that damage is exaggerated by hyperglycemia because it allows additional lactate to accumulate in the partially substrate-depleted tissue.

Cerebral metabolic changes in profound, insulin-induced hypoglycemia, and in the recovery period following glucose administration

Severe hypoglycemia was induced by insulin in lightly anaesthetized (70°o N2O) and artificially ventilated rats. Brain tissue was frozen in situ after spontaneous EEG potentials had disappeared for 5. 10. 15 or 30 min and cerebral cortex concentrations of labile organic phosphates, glycolytic metabolites, ammonia and amino acids were determined. In other experiments, recovery was induced by glucose injection at the end of the period of EEG silence.

Changes in the bioenergetic state of rat hippocampus during 2.5 min of ischemia, and prevention of cell damage by cyclosporin A in hyperglycemic subjects.

Abstract A recent study from this laboratory has shown that brief transient ischemia (2 min 30 s) in normo- and hyperglycemic rats leads to moderate neuronal necrosis in CA1 cells of the hippocampus, of equal density in the two groups. However, hyperglycemic animals failed to depolarize during the ischemia, nor did they show a decrease in extracellular calcium concentration. The present study was undertaken to study the metabolic correlates to these unexpected findings. Normoglycemic (plasma glucose ∼6 mM) and hyperglycemic (∼20 mM) rats were subjected to ischemic periods of 1 min and 2 min 15 s (2 min 30 s with freezing delay considered), and their brains were frozen in situ. Samples of dorsal hippocampus were dissected at –22°C and extracted for the measurement of phosphocreatine (PCr), creatine, ATP, ADP, AMP, glucose, glycogen, and lactate. Normoglycemic animals showed rapid depletion of PCr, ATP, glucose, and glycogen, and a rise in lactate content to 10–12 mM·kg–1 during the ischemia. Hyperglycemic animals displayed a more moderate rate of fall of PCr and ATP, with ATP values exceeding 50% of control after 2 min 30 s. Glycogen stores were largely maintained, but degradation of glucose somewhat enhanced the lactic acidosis. The results demonstrate that hyperglycemic rats maintained ATP at levels sufficient to prevent cell depolarization and calcium influx during the ischemic period. However, the metabolic perturbation observed must have been responsible for the delayed neuronal damage. We speculate that lowered ATP, increased inorganic P, and oxidative stress triggered a delayed mitochondrial permeability transition (MPT), which led to delayed neuronal necrosis. This assumption was supported by a second series of experiments in which CA1 damage in hyperglycemic rats was prevented by cyclosporin A, a virtually specific inhibitor of the MPT.

Phosphorylase a and Labile Metabolites During Anoxia: Correlation to Membrane Fluxes of K+ and Ca2+

Abstract: The objective of the present study was to explore mechanisms responsible for activation of ion conductances in the initial phases of brain ischemia, particularly for the early release of K+ that precedes massive cell depolarization, and rapid downhill fluxes of K+, Na+, Cl−, and Ca2+. As it has been speculated that a K+ conductance can be activated either by an increase in the free cytosolic calcium concentration (Ca2+i) or by a fall in ATP concentration, the question arises whether the early increase in extracellular K+ concentration (K+e) is preceded by a rise in Ca2+i and/or a fall in ATP content. In the present experiments, ischemia was induced in rats by cardiac arrest, the time courses of the rise in K+e and cellular depolarization were determined by microelectrodes, and the tissue was frozen in situ through the exposed dura for measurements of levels of labile metabolites, including adenine nucleotides and cyclic AMP (cAMP), after ischemic periods of 15, 30, 60, and 120 s. Conversion of phosphorylase b to a was assessed, because it depends, among other things, on changes in Ca2+i. The K+e value rose within a few seconds following induction of ischemia, but massive depolarization (which is accompanied by influx of calcium) did not occur until after ∼65 s. Activation of phosphorylase was observed already after 15 s and before glycogenolysis had begun. At that time, 3′,5′‐cAMP concentrations were unchanged, and total 5′‐AMP concentrations were only moderately increased. The results demonstrate that a K+ conductance is activated at a time when the overall ATP concentration remains at 95% of control values. If major compartmentation can be excluded, the results fail to demonstrate that an ATP‐activated K+ conductance is involved. In view of the early activation of phosphorylase, one may speculate that the triggering event is a rise in Ca2+i.

Changes of labile metabolites during anoxia in moderately hypo- and hyperthermic rats: correlation to membrane fluxes of K+.

The objective of this study was to assess the influence of temperature on the coupling among energy failure, depolarization, and ionic fluxes during anoxia. To that end, we induced anoxia by cardiac arrest in anesthetized rats maintained at a body temperature of either 34 degrees C or 40 degrees C, measured extracellular K+ concentration (K+e), and froze the neocortex through the exposed dura for measurements of phosphocreatine (PCr), creatine (Cr), ATP, ADP, and AMP, glucose, glycogen, pyruvate and lactate content after ischemic intervals of maximally 130 s. Free ADP (ADPf) concentrations were derived from the creatine kinase equilibrium. Hypothermia reduced the initial rate of rise in K+e, and delayed the terminal depolarization; however, both hypo- and hyperthermic animals showed massive loss of ion homeostasis at a K+e of 10-15 mM. The initial rate of rise in K+e did not correlate to changes in ATP, or ATP/ADPf ratio, suggesting that temperature changes per se may control the degree of activation of K+ conductances. The results clearly showed that, in both hyper- and hypothermic subjects, energy failure preceded the sudden activation of membrane conductances for ions. The results indicate that temperature primarily influences membrane permeability to ions like K+e (and Na+), and that cerebral energy state is secondarily affected. It is proposed that the higher rate of rise of K+e at high temperatures accelerates ATP hydrolysis primarily by enhancing metabolic rate in glial cells.

Cerebral Metabolic Changes During and Following Fluorothyl‐Induced Seizures in Ventilated Rats

Abstract: The objective of the present study was to assess metabolic changes in the neocortex and hippocampus of well‐oxygenated or moderately hypoxic rats in which fluorothyl‐induced seizures were sustained for 5 or 20 min, or which were allowed recovery periods of 5, 15, or 45 min following cessation of 20‐min seizure activity by withdrawal of the convulsant gas. Sustained fluorothyl‐induced seizures were found to cause metabolic alterations qualitatively and quantitatively similar to those previously observed with other commonly used convulsants. Thus, although the phosphorylation state of the adenine nucleotide pool remained only moderately perturbed, if at all, there were decreases in tissue concentrations of phosphocreatine and glycogen, and increases in those of cyclic AMP, lactate, and pyruvate, with a calculated fall in intracellular pH of about 0.15 units and a rise in the cytoplasmic NADH/NAD+ ratio. The enhanced metabolic rate was reflected in a marked reduction in the tissue‐to‐plasma glucose concentration ratio. Induced moderate hypoxia (arterial Po2 40–50 mm Hg) had no metabolic effect after 5 min of seizures but moderately increased lactate concentrations after 20 min (from about 10 to about 15 μmol ± g−1). On cessation of seizure discharge cyclic AMP and phosphocreatine concentrations normalized already within 5 min, whereas glycogen and lactate concentrations normalized more slowly. In the neocortex (but not the hippocampus) postepileptic tissue‐to‐plasma glucose concentration ratios rose above control, probably reflecting metabolic depression. The results suggest that intracellular pH promptly returned to control, and that postepileptic alkalosis developed. They also suggest that some elevation of the NADH/NAD+ ratio persisted even after 45 min of recovery.

Energy metabolism of mouse cerebral cortex during homocysteine convulsions.

Abstract Convulsions were induced in mice by the intraperitoneal injection of dl -homocysteine thiolactone HCl. Energy metabolism of the cerebral cortex was studied during both the clonic and tonic phases of convulsions, and 5 min after the main seizure attack. During both phases of convulsions a decreased level of P-creatine, ATP and glycogen, an unchanged level of glucose and an increased level of lactate, ADP and AMP were observed. The energy charge potential of the adenine nucleotide pool (ECP) was lowered. Since the accumulation of lactate was higher than could be accounted for by the decrease of glycogen, an approximately 3–5-fold increase of glucose uptake from blood has to be assumed. After 5 min of recovery, partial restoration of lactate and glycogen, increased levels of P-creatine and glucose and normal values of ECP were observed; the energy balance of the tissue was again re-established. During the preparoxysmal period only a slight increase of glucose was observed; the other metabolites studied were normal. Clinical manifestations of homocysteine convulsions could be prevented by a non-anaesthetic dose of Na phenobarbital or by glycine. Metabolic changes were completely prevented by Na phenobarbital; in the case of glycine protection, a slight increase of lactate and a decrease of glycogen were observed. The present results have demonstrated that the changes in energy metabolism of brain during homocysteine convulsions differ substantially from those observed previously during seizures induced by methionine sulphoximine (MSO) 5,9 . It therefore seems unlikely that homocysteine is an effective intermediary factor in MSO convulsions.

Does ischemia with reperfusion lead to oxidative damage to proteins in the brain

Recent results suggest that even relatively brief periods of ischemia in gerbils (10 min) lead to oxidative damage to brain proteins, reflected in an increased carbonyl content in the soluble protein fraction and a decreased glutamine synthetase (GS) activity. Since we failed to reproduce these findings in rats subjected to 15 min of transient ischemia, we explored whether oxidative damage to proteins could be observed after longer ischemic periods. To that end, one middle cerebral artery was occluded in rats for either 1 or 3 h, with recirculation periods of 0 min, 15 min, 1 h, and 6 h. Protein carbonyl content and GS activity were determined in focal and perifocal tissues and compared with values obtained in the same areas on the contralateral side. Ischemia, particularly of 3-h duration, followed by various reperfusion periods was accompanied by a significant (16–35%) decrease in the concentration of proteins of the soluble protein fraction. However, in no group was there an increased carbonyl content of the remaining proteins in this fraction. When expressed per milligram of protein, GS activity remained unchanged or rose somewhat. An inconsistent (and moderate) decrease in GS activity was present only if GS activity was expressed per milligram of wet tissue. The present findings, which fail to document oxidative damage to proteins following focal ischemia of 1- or 3-h duration, are thus radically different from those obtained in gerbils. The results suggest that appreciable species differences exist and raise the question of whether free radical–mediated oxidation of proteins is an invariable component of ischemic brain damage.

Changes of cyclic AMP and phosphorylase a in mouse cerebral cortex during seizures induced by 3-mercaptopropionic acid.

Abstract Cyclic AMP levels and glycogen phosphorylase a activity were investigated in the cerebral cortex of mice during seizures induced by 3-mercaptopropionic acid (MP). The onset of seizure was associated with a 3-fold increase of cyclic adenosine 3′,5′-monophosphate (c-AMP) and rapid activation of glycogen phosphorylase a (60% in the a form, i.e. to about double the values of the controls). Five minutes after the end of convulsions, both the levels of cyclic AMP and phosphorylase a activity were already restored to control values and remained unchanged during the subsequent 5 min. Sodium phenobarbital not only prevented clinical manifestations of MP convulsions, but also the changes in cyclic AMP content and phosphorylase activation observed during convulsions. These findings support the suggestion that cyclic AMP participates in the regulation of glycogen metabolism in the brain similarly as in other organs. The rise of cyclic AMP accompanying MP seizures was not influenced by the pretreatment of mice with theophylline which suggests that adenosine is probably not involved as a mediator of enhanced levels of cyclic AMP. The accumulation of cyclic AMP during MP seizures was markedly reduced by dl -propranolol (by about 20% and 80%, respectively, depending on the dose of propranolol used). This suggests that during MP seizures the possible release of endegenous catecholamines might interact with cyclic AMP generating system. Propranolol failed, however, to influence the activation of phosphorylase a. The failure of propranolol to protect activation of phosphorylase may be due to the fact that either a small increase in c-AMP (remaining after propranolol pretreatment, +20%) is sufficient for maximal activation of the enzyme or that other factors (e.g. Ca 2+ ) participate.

Changes in glycogen phosphorylase activity and glycogen levels of mouse cerebral cortex during convulsions induced by homocysteine.

Glycogen phosphorylase activity and glycogen levels were investigated in the cerebral cortex of mice of two different strains under the influence of homocysteine. Control levels of glycogen and total phosphorylase activity (i. e. activity in the presence of 1 mM‐AMP) were higher in the inbred strain A, whereas a higher proportion of phosphorylase in its active form (activity without 5′‐AMP) was obtained in the ICR strain (probably due to slower fixation of brain in this strain). Changes occurring after the administration of homocysteine were similar in both strains. With the onset of first clonic seizures a marked increase of phosphorylase a occurred (increase 99 per cent in strain A and 46.5 per cent in ICR, respectively). During the latter phase of tonic seizures active phosphorylase a did not significantly differ from control values. Five minutes after the end of a tonic seizure, i. e. when partial recovery could already be observed, a marked decrease of active phosphorylase a in comparison with control values, was evident (decrease against control values of 45.5 per cent in strain A and 30.5 per cent in ICR, respectively). The total phosphorylase activity was not affected in strain A, whereas a slight increase during clonic seizures was seen in the ICR strain. In accordance with the enhanced activation of phosphorylase at the onset of clonic seizures, a marked decrease in glycogen levels (35‐50 per cent) was observed in both strains of mice. This decrease persisted even during the 5 min recovery period. When seizures were prevented by Na phenobarbital or glycine, the activation of phosphorylase was either completely prevented (by a non‐anaesthetic dose of phenobarbital) or reduced (by glycine). The present results have demonstrated that changes in glycogen metabolism occurring during homocysteine seizures differ distinctly from those previously found during seizures induced by methionine sulphoximine, a substance structurally related to homocysteine.