Ken-ichiro Katsura

Coupling Among Energy Failure, Loss of Ion Homeostasis, and Phospholipase A2 and C Activation During Ischemia

Abstract— The objective of the present experiments was to correlate changes in cellular energy metabolism, dissipative ion fluxes, and lipolysis during the first 90 s of ischemia and, hence, to establish whether phospholipase A2or phospholipase C is responsible for the early accumulation of phospholipid hydrolysis products. Ischemia was induced for 15–90 s in rats, extracellular K+ (K+e) was recorded, and neocortex was frozen in situ for measurements of labile tissue metabolites, free fatty acids, and diacylglycerides. Ischemia of 15‐and 30‐s duration gave rise to a decrease in phosphocreatine concentration and a decline in the ATP/free ADP ratio. Although these changes were accompanied by an activation of K+ conductances, there were no changes in free fatty acids until after 60s, when free arachidonic acid accumulated. An increase in other free fatty acids and in total diacylglyceride content did not occur until after anoxic depolarization. The results demonstrate that the early functional changes, such as activation of K+ conductances, are unrelated to changes in lipids or lipid mediators. They furthermore suggest that the initial lipolysis occurs via both phospholipase A2 and phospholipase C, which are activated when membrane depolarization leads to influx of calcium into cells.

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.

Extra- and Intracellular pH in the Brain During Ischaemia, Related to Tissue Lactate Content in Normo- and Hypercapnic rats.

The objective of the present study was to assess the relationship between the amount of lactate accumulated during complete ischaemia and the ensuing changes in extra‐ and intracellular pH (pHe and pHi respectively). The preischaemic plasma glucose concentration of anaesthetized rats was varied by administration of glucose or insulin, pHe was determined in neocortex with ion‐sensitive microelectrodes, and tissue lactate and CO2 contents were measured, tissue CO2 tension being known from separate experiments. The experiments were carried out in both normocapnic [arterial CO2 tension (PaCO2) ‐40 mm Hg] and hypercapnic (PaCO2 ‐80 mm Hg) animals. Irrespective of the preischaemic CO2 tension, δpHe was linearly related to tissue lactate content. Depending on the preischaemic glucose concentration, δpHe varied from <0.4 to >1.4 units. The results thus fail to confirm previous results that the changes in pHe describe two plateau functions (δ pHe‐0.5 and 1.1, respectively), with a transition zone at tissue lactate contents of 17–20 mmol kg−1. Changes in pH; given in this study are based on the assumption of a uniform intracellular space. The pH, changed from a normal value of ‐7.0 to 6.5, 6.1 and 5.8 at tissue lactate contents of 10, 20 and 30 mmol kg‐1. The intrinsic (non‐bicarbonate) buffer capacity, derived from these figures, was 23 mmol kg −1 pH−1. Some differences in pH and in HCO3− concentration between extra‐ and intracellular fluids persisted in the ischaemic tissue. These differences were probably caused by a persisting membrane potential in the ischaemic cells.

Critical values for plasma glucose in aggravating ischaemic brain damage: correlation to extracellular pH

The objective of the present experiments was to characterize conditions under which pre-ischaemic hyperglycaemia aggravates brain damage following transient forebrain ischaemia. Specifically, we wished to explore whether accentuated damage is a threshold function of plasma glucose concentration or pH, as assessed by measurements of extracellular pH (pHe). Forebrain ischaemia of 10 min duration was induced in rats at varying degrees of hyperglycaemia, with continuous measurements of pHe, and the animals were allowed to survive for 7 days before histopathological evaluation of the density and distribution of brain damage. Ischaemic brain damage appeared as a threshold function of plasma glucose concentration. At values of 4-6 mM virtually no damage was observed in any other structure than the CA1 sector of the hippocampus and, even in that structure, damage was variable. At glucose concentrations of 8-12 mM moderate damage was observed infrequently in caudoputamen, parietal cortex, and thalamus. At values above 12 mM, damage increased dramatically in these areas, and additional structures were recruited in the damage process (cingulate cortex, the CA3 sector of the hippocampus, and substantia nigra). Measurements of pHe in parietal cortex showed a threshold for seizure induction at values of 6.4-6.5, probably corresponding to intracellular pH values of 6.2-6.3. The threshold for aggravation of histopathological damage was similar. It is concluded that a moderate increase in plasma glucose in the threshold range predisposes the tissue to aggravated damage, probably by activating biochemical reactions or pathophysiological events with a steep pH dependence.

The influence of plasma glucose concentrations on ischemic brain damage is a threshold function.

To investigate whether aggravation of damage in hyperglycemic subjects is a continuous function of changes in intra- and extracellular pH during ischemia or whether there is a threshold value, preischemic plasma glucose was varied from 8.3-20.0 mM. 10 min forebrain ischemia was induced. The results showed that no animal with plasma glucose of < 13 mM developed seizures, and that all animals with glucose of > 16 mM died in status epilepticus. Half of the animals with plasma glucose in the range of 13-16 mM showed seizures and 50% of these died. In surviving animals, histological brain damage occurred in the hippocampal CA3 sector, cingulate cortex, thalamic nuclei and substantia nigra, structures normally not injured by 10 min ischemia. The data demonstrate that there is a glucose threshold of 10-13 mM, above which seizures develop and additional damage appears, and another one (> 16 mM), above which seizures are invariably fatal.

Extracellular pH in the brain during ischemia: relationship to the severity of lactic acidosis.

On the basis of data showing a bimodal distribution of values for extracellular pH (pHe), and a discontinuous ΔPco2/Δlactate relationship, Kraig et al. (1986) proposed that H+ is grossly compartmentalized between neurons and glia in the ischemic brain. We measured ΔpHe during ischemia, varying ischemic lactate contents between 9 and 38 mmol kg−1. No bimodal distribution was found, but ΔpHe varied linearly with lactate content. Because we have also failed to record a discontinuous ΔPco2/Δlactate relationship, we conclude that major compartmentation of H+ does not occur during ischemia.

Chapter 3 Acidosis-related brain damage

Publisher Summary This chapter updates information on the coupling among hyperglycemia, intra and extracellular acidosis and brain damage because of ischemia or hypoxia, and discusses cellular and molecular mechanisms that may be involved. When ischemia is complicated by excessive acidosis, the ischemic damage encompasses post-ischemic seizures, edema, and pannecrosis. The cellular and molecular mechanisms responsible for these alterations have not been adequately defined. However, it seems likely that the acidosis causes damage to inhibitory GABAergic cells by raising Ca i 2+ to levels, which will overload the buffering systems and cause cell death, thereby explaining the proclivity to post-ischemic seizure discharge. The rapidly evolving damage following long periods of ischemia is probably caused by several adverse effects of a raised H + activity: inhibition of Na + /H + exchange and lactate – oxidation, inhibition of mitochondria1 respiration, and acceleration of coupled Na + /H + and Cl – /HCO 3 – exchange. However, an important factor may be a lingering rise in Ca i 2+ in cells whose pH i is reduced over a longer period, predisposing to Ca 2+ -related damage. The molecular mechanisms underlying delayed acidosis-related damage probably comprise Fe 2+ and NO · -related production of free radicals that have the microvessels as their main target.

Hypoglycemia-Elicited Immediate Early Gene Expression in Neurons and Glia of the Hippocampus: Novel Patterns of FOS, JUN, and KROX Expression following Excitotoxic Injury

In the hippocampus there is a graded vulnerability of neuronal subpopulations to hypoglycemia-induced degeneration, most likely due to excitotoxic activation of glutamate receptors. The present study was conducted to investigate whether the induction of transcription factors of the immediate early gene (IEG) family after hypoglycemia reflects these different grades of neuronal vulnerability. We studied the expression profile of seven IEG-coded proteins in the rat hippocampus following severe insulin-induced hypoglycemia with 30 min of EEG isoelectricity and various survival periods for up to 42 h after glucose replenishment. Immunocytochemistry was performed on vibratome sections with specific polyclonal antisera directed against c-FOS, FOS B, c-JUN, JUN B, JUN D, KROX-24, and KROX-20. To unequivocally define the type of glial cells showing IEG induction, we investigated coexpression of c-FOS and glial marker proteins (glial fibrillary acid protein [GFAP], OX-42) by confocal laser scanning microscopy. Up to 3 h after glucose replenishment, differential temporospatial induction of IEG-coded transcription factors of the FOS, JUN and KROX families were observed in moderately injured neuronal subpopulations, including the majority of dentate granule cells and CA3 neurons. At later time points, however, a delayed and persistent c-JUN expression was found in severely, but reversibly, injured CA1 neurons and in neurons in the immediate vicinity of irreversibly damaged neurons in the crest of the dentate gyrus. Similar to the results with experimental models of central and peripheral axotomy, selective c-JUN induction in these neurons may represent an initial event in the regeneration process of sublethally injured neurons. In contrast to other models of excitotoxic injury such as ischemia and epilepsy, marked glial c-FOS expression was restricted to astrocytes, as assessed by confocal laser scanning microscopy.

Tissue Pco2 in Brain Ischemia Related to Lactate Content in Normo- and Hypercapnic Rats

The amount of lactate formed during ischemia determines the rise in tissue Pco2 (Ptco2). Conflicting results exist on the relationship between lactate and Ptco2. The objective of this study was to settle this issue. We varied the preischemic plasma glucose concentration of normo- and hypercapnic rats, assessed tissue lactate and total CO2 contents, and determined the Pco2/lactate relationship over the lactate range 2–40 mmol kg−1. The results showed that whatever the equilibration time, the Pco2/lactate relationship was linear. The results obtained could be reproduced by a theoretical buffer system that mimics the buffering behavior of intracellular fluid. Our results bear on the question of whether compartmentation of H+ occurs during ischemia, with glial cells becoming more acid than neurons. A discontinuous Pco2/lactate relationship, with a constant Pco2 above a certain lactate content, would support this contention. Since our results demonstrate a linear relationship between lactate and Pco2 over the lactate range 2–40 mmol kg−1, they considerably weaken any argument for gross compartmentation of H+.