Tibor Kristián

Acidosis Induced by Hypercapnia Exaggerates Ischemic Brain Damage

Although preischemic hyperglycemia is known to aggravate damage due to transient ischemia, it is a matter of controversy whether or not this is a result of the exaggerated acidosis. It has recently been reported that although tissue acidosis of a comparable severity could be induced in normoglycemic dogs by an excessive rise in arterial CO2 tension, short-term functional recovery was improved, rather than compromised. In the present experiments we induced excessive hypercapnia (Paco2, ∼300 mm Hg) in normoglycemic rats before inducing forebrain ischemia of 10-min duration. This reduced the brain extracellular pH to values normally encountered in hyperglycemic rats subjected to ischemia. The events induced by hypercapnia clearly enhanced ischemic brain damage, as assessed histologically after 7 days of recovery. We hypothesize that the decisive event was an exaggerated decrease in extra- and intracellular pH and that the results thus demonstrate an adverse effect of acidosis. However, since postischemic seizures did not occur in the hypercapnic ischemic rats, the results also demonstrate that changes in intra-extracellular pH and bicarbonate concentrations modulated ischemic damage in an unexpected way.

Molecular Mechanisms of Acidosis-Mediated Damage

The present article is concerned with mechanisms which are responsible for the exaggerated brain damage observed in hyperglycemic animals subjected to transient global or forebrain ischemia. Since hyperglycemia enchances the production of lactate plus H+ during ischemia, it seems likely that aggravation of damage is due to exaggerated intra- and extracellular acidosis. This contention is supported by results showing a detrimental effect of extreme hypercapnia in normoglycemic rats subjected to transient ischemia or to hypoglycemic coma. Enhanced acidosis may exaggerate ischemic damage by one of three mechanisms: (i) accelerating free radical production via H(+)-dependent reactions, some of which are catalyzed by iron released from protein bindings by a lowering of pH, (ii) by perturbing the intracellular signal transduction pathway, leading to changes in gene expression or protein synthesis, or (iii) by activating endonucleases which cause DNA fragmentation. While activation of endonucleases must affect the nucleus, the targets of free radical attack are not known. Microvessels are considered likely targets of such attack in sustained ischemia and in trauma; however, enhanced acidosis is not known to aggravate microvascular dysfunction, or to induce inflammatory responses at the endothelial-blood interface. A more likely target is the mitochondrion. Thus, if the ischemia is of long duration (30 min) hyperglycemia triggers rapidly developing mitochondrial failure. It is speculated that this is because free radicals damage components of the respiratory chain, leading to a secondary deterioration of oxidative phosphorylation.

The influence of pH on cellular calcium influx during ischemia.

The objective of this study was to explore how alterations in tissue pH during ischemia influence cell calcium uptake, as this is reflected in the extracellular calcium concentration (Ca2+e). Variations in pH were achieved by making animals hypo-, normo- or hyperglycemic prior to cardiac arrest ischemia or by increasing preischemic PCO2 in normoglycemic animals. For comparison, the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine maleate (MK-801) was given prior to induction of ischemia. In some experiments the effect of acidosis on K+ efflux and Na+ influx were studied as well. In hypoglycemic subjects, the reduction of Ca2+e during ischemia was very rapid, 90% of the reduction occurring within 4.7 s. Normoglycemic animals showed a slower rate of reduction of Ca2+e. Hyperglycemic animals displayed an even slower rate of reduction and a biphasic response in which the initial, faster influx of Ca2+ was followed by a conspicuously slow one. This second phase led to a very gradual decrease in Ca2+e, a stable level being reached first after 6-7 min. This marked delay in calcium influx during ischemia was very similar in hypercapnic animals, who showed an extracellular pH during ischemia as low as hyperglycemic subjects. The effect of acidosis was duplicated by MK-801, suggesting that low pH reduces calcium influx by blocking NMDA-gated ion channels.

Influence of acid-base changes on the intracellular calcium concentration of neurons in primary culture.

The influence of changes in intra- and extracellular pH (pHi and pHe, respectively) on the cytosolic, free calcium concentration ([Ca2+]i) of neocortical neurons was studied by microspectrofluorometric techniques and the fluorophore fura-2. When, at constant pHe, pHi was lowered with the NH4Cl prepulse technique, or by a transient increase in CO2 tension, [Ca2+]i invariably increased, the magnitude of the rise being proportional to ΔpHi. Since similar results were obtained in Ca2+-free solutions, the results suggest that the rise in [Ca2+]i was due to calcium release from intracellular stores. The initial alkaline transient during NH4Cl exposure was associated with a rise in [Ca2+]i. However, this rise seemed to reflect influx of Ca2+ from the external solution. Thus, in Ca2+-free solution NH4Cl exposure led to a decrease in [Ca2+]i. This result and others suggest that, at constant pHe, intracellular alkalosis reduces [Ca2+]i, probably by enhancing sequestration of calcium. When cells were exposed to a CO2 transient at reduced pHe, Ca2+ rose initially but then fell, often below basal values. Similar results were obtained when extracellular HCO3-concentration was reduced at constant CO2 tension. Unexpectedly, such results were obtained only in Ca2+-containing solutions. In Ca2+-free solutions, acidosis always raised [Ca2+]i. It is suggested that a lowering of pHe stimulates extrusion of Ca2+ by ATP-driven Ca2+/2H+ antiport.

Chapter 2 Changes in Ionic Fluxes During Cerebral Ischaemia

Publisher Summary The flux of ions through ion channels is passive, requiring no metabolic energy. The direction of this flux and the equilibrium are determined by the electrochemical driving force across the membrane. This driving force is determined by two factors: the electrical potential difference (membrane potential) and the concentration gradient of the permeate ions across the membrane. Thus, if the membrane potential is identical with the equilibrium potential (E P ) for a given ion, a condition when the electrical and chemical forces are equal, there is no net ion flux across the cell membrane, even if the channels conducting this ion are open. Under all conditions, the membrane potential is close to the E P , for the most permeable ion(s). A dramatic change in ionic fluxes occurs secondary to bioenergetic failure during ischemia and hypoglycemia. Ischemia can be divided into two major types: global or forebrain ischemia of the cardiac arrest type and focal ischemia of the stroke type. Because ion gradients across nerve and glial cell membranes are upheld at the expense of energy in the form of ATP, energy failure due to ischemia leads to the dissipation of ionic gradients. Ischemia also causes a gradual fall in extra- and intracellular pH due to the activation of glycolysis, with production of lactate and hydrogen ion (H + ).

Effects of Preischemic Hyperglycemia on Brain Damage Incurred by Rats Subjected to 2.5 or5 Minutes of Forebrain Ischemia

BACKGROUND AND PURPOSE The objective of this study was to explore whether preischemic hyperglycemia, which is known to aggravate brain damage due to transient global or forebrain ischemia of intermediate duration (10 to 20 minutes), increases the density of selective neuronal necrosis, as observed primarily in the CA1 sector of the hippocampus after brief periods of forebrain ischemia in rats (2.5 and 5 minutes). METHODS Anesthetized rats were subjected to two-vessel forebrain ischemia of 2.5- or 5-minute duration. Normoglycemic or hyperglycemic rats were either allowed a recovery period of 7 days for histopathological evaluation of neuronal necrosis in the hippocampus, isocortex, thalamus, and substantia nigra or were used for recording of extracellular concentrations of Ca2+ ([Ca2+]c), K+, or H+, together with the direct current (DC) potential. RESULTS Ischemia of 2.5- or 5-minute duration gave rise to similar damage in the CA1 sector of the hippocampus in normoglycemic and hyperglycemic groups (10% to 15% and 20% to 30% of the total population, respectively). However, in hyperglycemic animals subjected to 2.5 minutes of ischemia, CA1 neurons never depolarized and [Ca2+]c did not decrease. In the 5-minute groups, the total period of depolarization was 2 to 3 minutes shorter in hyperglycemic than in normoglycemic groups. This fact and results showing neocortical, thalamic, and substantia nigra damage in hyperglycemic animals after 5 minutes of ischemia demonstrate that although hyperglycemia delays the onset of ischemic depolarization and hastens repolarization and extrusion of Ca2+, it aggravates neuronal damage due to ischemia. CONCLUSIONS These results reinforce the concept that hyperglycemia exaggerates brain damage due to transient ischemia and prove that this exaggeration is observed at the neuronal level. The results also suggest that the concept of the duration of an ischemic transient should be qualified, particularly if ischemia is brief, ie. < 10 minutes in duration.

Release of mitochondrial aspartate aminotransferase (mAST) following transient focal cerebral ischemia suggests the opening of a mitochondrial permeability transition pore

The present experiments were undertaken to study if ischemia and reperfusion in the brain are accompanied by a mitochondrial permeability transition (MPT), leading to the release of mitochondrial proteins into the cytosol. The protein studied was the mitochondrial isoform of aspartate aminotransferase (mAST). In vitro experiments showed that isolated brain mitochondria exposed to calcium (50 μM) and phosphate (0.5 mM) displayed rapidly developing swelling, as evidenced by a change in light scattering at 540 nm. The swelling which could be reversal by chelation of external calcium, was associated with release of mAST. Focal ischemia of 2 h duration was induced by occlusion of one middle cerebral artery (MCA) by an intraluminal filament technique. The ischemia gave rise to a marked decrease in respiratory control ratio (RCR) of mitochondria in the homogenate. The RCR recovered partly after 1 h of recirculation, but decrease again after 4 h. The mAST in the cytosolic fraction did not change at the end of the ischemia, but increased significantly after 1 h of recirculation in the focus of the lesion, but not in the penumbra. Little further change occurred thereafter (4 h). It is concluded that in densely ischemic areas, recirculation yields rapidly developing dysfunction of the inner mitochondrial membrane, with release of mitochondrial proteins.

Induced spreading depressions in energy-compromised neocortical tissue: calcium transients and histopathological correlates.

Mechanisms causing gradual recruitment of damaged cells in the penumbra zone around the core of a focal ischaemic lesion may encompass irregularly occurring depolarization waves of the spreading depression (SD) type, each leading to transient loading of cells with calcium. It has been speculated that, when elicited in an underperfused or otherwise energy-compromised tissue, such depolarization waves lead to cell damage. We assessed under what conditions the calcium transients during KCl-induced SDs are prolonged, and explored if marked prolongation of the transients leads to brain damage. Cerebral blood flow (CBF) was reduced by marked hypocapnia. Tissue oxygenation was reduced by arterial hypoxia, without or with unilateral carotid artery occlusion, or by occlusion of the carotid arteries in normoxic, anaesthetized rats. In all animals the DC potential and extracellular calcium concentration (Ca2+e) were measured before and during a series of SDs. The animals were recovered for histopathological assessment. Hypoxia alone (Pao2, 32.5 +/- 3.8 mmHg) increased mean and total depolarization times, but repeated SDs elicited over 1.7 (+/-0.4) h failed to induce cell damage. Unilateral carotid artery occlusion further prolonged the SD waves but, in spite of total depolarization times of up to 40 min during 2 h, only two out of seven animals showed damage, localized to caudoputamen and parietal cortex, as well as to the subiculum, CA1 and CA3 sectors of the hippocampus. Bilateral carotid artery occlusion was associated with the most pronounced prolongation of the DC potential shifts and Ca2+ transients, with total depolarization times of up to 70 min. In spite of this, only four out of 13 animals showed brain damage and in two of these the damage was contralateral. The results justify modification of the hypothesis stating that SD-like depolarizations in the perifocal penumbra zone per se is what leads to gradual recruitment of such tissues in the infarction process. It is suggested that additional factors are required, such as a larger reduction in CBF, or the proximity of cells at risk to necrotic tissue.

Brain Calcium Metabolism in Hypoglycemic Coma

The present experiments were designed to provide information on brain calcium metabolism during hypoglycemic coma. We specifically wished to evaluate changes in extracellular calcium concentration (Ca2+e) during prolonged hypoglycemic coma and recovery and to assess whether Ca2+e falls to similar values during hypoglycemia and ischemia. To that end, Ca2+e and K+e in neocortical tissue were recorded by ion-sensitive microelectrodes during hypoglycemic coma of 30 min duration and during 15 min of recovery. Cardiac arrest ischemia was induced either at the end of the period of hypoglycemia or after 15 min of recovery. Hypoglycemic coma, as reflected by a DC potential shift and by cellular release of K+, was accompanied by a sustained decrease in Ca2+e from ∼1.2 to ∼0.02 mM, i.e., to ∼1% of control. Infusion of glucose was followed by a biphasic recovery of Ca2+e, starting within 2 min of infusion. During the first phase, completed within the initial 3–4 min, Ca2+e rose to about 25% of control. During the second phase, Ca2+e slowly increased toward normal within 25–30 min. Ischemia, when induced at the end of the period of hypoglycemia, was accompanied by a rise in Ca2+e to about 0.1 mM, i.e., about 10% of control. A similar value was recorded when ischemia was induced after 15 min of recovery following a 30-min hypoglycemic coma. Although the present results do not give information on Ca2+i during hypoglycemic coma, it is tempting to conclude that partial preservation of the nucleoside triphosphate stores, and absence of acidosis, allow some binding and sequestration of the calcium entering the cell. Such binding and sequestration may explain the pronounced biphasic nature of the recovery curves for Ca2+e. Thus, if one assumes that the rapid increase in Ca2+e represents extrusion of available “free” intracellular Ca2+, one can envisage that the slow phase of recovery represents calcium that is slowly extruded following its release from the binding and sequestration stores.

Regulation of intra- and extracellular pH in the rat brain in acute hypercapnia: a re-appraisal

Recent results have demonstrated that intracellular pH (pHi) in nerve and glial cells is not regulated back to normal during CO2 exposure if extracellular pH (pHe) is reduced. This raises the question about regulation of pHi and pHe in vivo. In order to successively reduce pHe we exposed animals to incremental increases in CO2 tension (11, 27.5, 42.5%) and studied regulation of pHi during the first 90 min of hypercapnia. Extracellular pH, as well as Na+, K+, and Cl- concentrations, were also measured, as were whole tissue contents of Na+, K+, and Cl-. At all CO2 tensions studied, pHe slowly increased during CO2 exposure. In animals breathing 11% CO2 (delta pHe approximately 0.2 units), pHi increased slowly. However, in animals exposed to 27.5% CO2 or 42.5% CO2 (delta pHe > 0.4 units), no regulation of pHi was observed. Extracellular HCO3- concentrations increased substantially already during the first 15 min of hypercapnia (not significant in animals breathing 42.5% CO2), and then gradually rose. These increases were accompanied by a decrease in Cl- and an increase in Na+ concentration, K+ concentration remaining constant. The total tissue content of these ions remained constant, suggesting that extracellular HCO3- concentration increases by Cl-/HCO3- antiport and/or by Na+.2HCO3- symport, the HCO3- emanating from intracellular sources. The results challenge the dogma of the supremacy of mechanisms regulating pHi, and suggest that brain cells, possibly astrocytes, regulate pHe at the expense of their own pH homeostasis. By inference, we further conclude that regulation of pHi normally occurs only if pHe is first regulated back close to normal value.