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Effects of Mild Hypothermia on Metabolic Disturbances in Fetal Hippocampal Slices After Oxygen/Glucose Deprivation Depend on Depth and Time Delay of Cooling

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Abstract

Objective:There is increasing evidence from animal experiments that mild hypothermia induced during or after cerebral ischemia might protect the immature brain from neuronal cell damage. However, the exact interrelation between the postischemic time delay and the degree of mild hypothermia by which to achieve neuroprotective effects on ischemic insults of different severity has not yet been elucidated systematically. To determine optimal neuroprotection, we studied the intention between these variables in a recently modified hippocampal slice model.

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Children who suffer from perinatal brain injury often deal with the dramatic consequences of this misfortune for the rest of their lives. Despite the severe clinical and socioeconomic significance, no effective clinical strategies have yet been developed to counteract this condition. As shown in recent studies, perinatal brain injury is usually brought about by cerebral ischemia, cerebral hemorrhage, or an ascending intrauterine infection. This review focuses on the pathophysiologic pathways activated by these insults and describes neuroprotective strategies that can be derived from these mechanisms. Fetal cerebral ischemia causes an acute breakdown of neuronal membrane potential followed by the release of excitatory amino acids such as glutamate and aspartate. Glutamate binds to postsynaptically located glutamate receptors that regulate calcium channels. The resulting calcium influx activates proteases, lipases, and endonucleases, which in turn destroy the cellular skeleton. A second wave of neuronal cell damage occurs during the reperfusion phase. This cell damage is thought to be caused by the postischemic release of oxygen radicals, synthesis of nitric oxide, inflammatory reactions, and an imbalance between the excitatory and inhibitory neurotransmitter systems. Furthermore, secondary neuronal cell damage may be brought about in part by induction of a cellular suicide program known as apoptosis. Recent studies have shown that inflammatory reactions not only aggravate secondary neuronal damage after cerebral ischemia, but may also injure the immature brain directly. This damage may be mediated by cardiovascular effects of endotoxins leading to cerebral hypoperfusion and by activation of apoptotic pathways in oligodendrocyte progenitors through the release of proinflammatory cytokines. Periventricular or intraventricular hemorrhage (PIVH) is a typical lesion of the immature brain. The inability of preterm fetuses to redistribute cardiac output in favor of the central organs and their lack of cerebral autoregulation may cause significant fluctuations in cerebral blood flow when oxygen is in short supply. Disruption of the thin-walled blood vessels in the germinal matrix with subsequent cerebral hemorrhage is often the inevitable result and is at times associated with cerebral hemorrhagic infarction. Knowledge of these pathophysiologic mechanisms has enabled scientists do develop new therapeutic strategies, which have been shown to be neuroprotective in animal experiments. The potential of such therapies is discussed here, particularly the promising effects of postischemic induction of cerebral hypothermia, the application of the calcium-antagonist flunarizine, and the administration of magnesium.
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To investigate the effects of hypothermia on the rate of change and degree of recovery of brain adenosine triphosphate and phosphocreatine concentrations and intracellular pH, we have developed a model that allows phosphorus nuclear magnetic resonance spectroscopy of the intact piglet brain during circulatory arrest. Three groups of piglets were studied. Three control animals underwent cardiopulmonary bypass at normothermia for 1 hour; five group 1 animals underwent bypass at a brain temperature of 15 degrees C, followed by a period of circulatory arrest such that adenosine triphosphate was absent for 21 minutes, followed by 1 hour of reperfusion; and five group 2 animals underwent bypass at a brain temperature of 37 degrees C, followed by a period of circulatory arrest such that adenosine triphosphate was absent for 21 minutes, followed by reperfusion for 1 hour. Control animals showed no significant metabolic effects of bypass. Group 1 animals showed a slower decay of the adenosine triphosphate and phosphocreatine concentrations than group 2 animals, consistent with a lower metabolic rate, and had a higher pH at the onset of ischemia. Recovery of the adenosine triphosphate concentration was significantly better in group 1 animals (95%) than in group 2 animals (30%) (p less than 0.02), and recovery of the phosphocreatine concentration was also better in group 1 animals (93%) than in group 2 animals (32%) (p less than 0.02). Intracellular pH recovered in group 1 animals, but not in group 2 animals. Regional biochemical assays of metabolites performed in the group 2 piglets and in five pilot piglets exposed to deep hypothermia generally confirmed the spectroscopic findings but demonstrated considerable regional variation, specially in the group 2 piglets' brains. We conclude that hypothermia exerts a protective effect on the piglet brain during global ischemia even after the adenosine triphosphate pool has been completely depleted.
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The alterations in tissue metabolism induced by hypothermic cardiopulmonary bypass are not completely known. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to determine the effect of hypothermic cardiopulmonary bypass on energy states and intracellular pH of the heart and brain. Sheep were instrumented for cardiopulmonary bypass and had a radiofrequency coil placed over either the heart or skull. The animals were placed in a 4.7-T magnet at 37 degrees C and spectra obtained. The animals were cooled on cardiopulmonary bypass to either 26 degrees C (n = 17) or 18 degrees C (n = 14) for brain studies and to 26 degrees C (n = 12) for heart studies. Hypothermia increased the phosphocreatine/adenosine triphosphate ratio in the heart (2.38 +/- 0.23 versus 3.18 +/- 0.37, 37 degrees versus 26 degrees C, respectively, p = 0.03). The brain phosphocreatine/adenosine triphosphate ratio increased from 1.70 +/- 0.09 at 37 degrees C to 2.00 +/- 0.12 at 26 degrees C (p = 0.009) and 2.10 +/- 0.07 at 18 degrees C (p = 0.0001). Intracellular pH increased during hypothermia (heart: 7.05 +/- 0.02 to 7.18 +/- 0.02, 37 degrees versus 26 degrees C, p = 0.0001; and brain: 7.07 +/- 0.02 versus 7.32 +/- 0.02, 37 degrees versus 18 degrees C, p = 0.0001). The adenosine triphosphate resonance position is known to be sensitive to magnesium binding as well as temperature and was shifted upfield (p less than 0.01) in both the heart and brain. This effect could be totally explained by the temperature dependence of this process.(ABSTRACT TRUNCATED AT 250 WORDS)
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Regional cerebral protein synthesis was investigated in the Mongolian gerbil during recovery from forebrain ischemia produced by bilateral common carotid artery occlusion for 5 min. At various recirculation periods up to 72 h animals received a single dose of L-(3,5-3H)tyrosine and were killed 30 min later. Brains were processed for autoradiography using the stripping film technique. During the initial 30 min of recirculation autoradiographs revealed an almost complete inhibition of protein synthesis in all forebrain structures with the exception of the medio-dorsal thalamic nuclei. Between 30 min and 12 h of recirculation amino acid incorporation was completely restored in neurons of the cerebral cortex, basal ganglia, hippocampal CA3 and CA4 zones and the dentate gyrus. In CA1, early (90-min postischemia) and progressive recovery of a few irregularly dispersed neurons was observed, but the vast majority of pyramidal cells never regained their normal biosynthetic activity. Between 3 and 6 h of recirculation CA1 neurons showed faint labeling, followed by a secondary deterioration resulting in complete lack of incorporation within 12 h after restoration of blood flow. Autoradiographs at all subsequent time points exhibited persistent inhibition of protein synthesis in CA1 until neuronal necrosis occurred 2-3 days later. Thus, in contrast to ischemia-resistant cell populations with rapid progressive and complete restoration of protein synthesis, hippocampal neurons undergoing delayed necrosis are characterized by an early incomplete recovery immediately followed by a secondary persistent inhibition.
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This chapter describes the cerebral protein synthesis rates (CPSR) in detail for normal rat and gerbil brains. The in vitro results are used as a supplement to correct the end-point measurements of specific activities in the absence of knowledge about their time course during the 45 minutes of incorporation. This approach is applied to the study of regional cerebral protein synthesis in two models of cerebral ischemia. Functional and morphological investigations have revealed that irreversible neuronal damage may develop after as little as five minutes' ischemia in selectively vulnerable areas, whereas recovery from ischemia seems to be possible in other regions of the brain after ischemia as long as one hour. There are two series of experiments: a short-lasting ischemia induced by bilateral carotid occlusion in gerbils, and a prolonged ischemia induced by intrathoracal clamping of the innominate, subclavian and mammary arteries in monkeys, are presented.
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The energy charge of the adenylate system, half of the average number of anhydride-bound phosphate groups per adenine moiety, has been proposed as a metabolic regulatory parameter. For several reactions that participate in biosynthesis or other adenosine triphosphate utilizing sequences, plots of enzyme activity against energy charge have positive slopes that increase with charge; thus these curves (type U) are concave upward and steep in the region of high charge. End-product feedback inhibition of the type demonstrated for many biosynthetic regulatory enzymes must be reflected in a decrease in the slope of such curves on the addition of the end product. The area between the curve representing absence of end product and that representing a saturating level of it should indicate the operational range of the regulatory enzyme. Within this range, if either end-product concentration or energy charge is constant the enzyme will respond only to variation in the other; but it may be expected that both parameters affect the behavior of the enzyme in the intact cell. For several reactions that participate in adenosine triphosphate regenerating sequences, plots of enzyme activity against energy charge have negative slopes that increase with charge; thus these curves (type R) are concave downward and steep in the region of high charge. Such sequences also supply primary metabolic intermediates needed as starting points in biosyntheses, and in some cases have been shown to be regulated also by the concentration of one or more of these intermediates. Inhibition of this type should be reflected in an increase in the negative slope of such curves on addition of the regulatory metabolite. As in the case of type U curves, a regulatory area will exist between the curves representing zero concentration and saturating concentration of the modifying metabolite. Experimental examples of both types of pattern are provided in the following two papers. It is proposed that the overlap of such regulatory type R and type U patterns illustrates graphically some of the ways in which energy charge and the concentrations of primary intermediates and of biosynthetic end products interact to stabilize the energy charge and to adjust the partitioning of substrates among competing metabolic functions in response to changing metabolic situations in the cell.
Article
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Article
Perturbants of the endoplasmic reticulum (ER), including Ca(2+)-mobilizing agents, provoke a rapid suppression of translational initiation in conjunction with an increased phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF)-2. Depletion of ER Ca2+ stores was found to signal the activation of a specific eIF-2 alpha kinase. Analysis of extracts derived from cultured cells that had been pretreated with Ca2+ ionophore A23187 or thapsigargin revealed a 2-3-fold increase in eIF-2 alpha kinase activity without detectable changes in eIF-2 alpha phosphatase activity. A peptide of 65-68 kDa, which was phosphorylated concurrently with eIF-2 alpha in extracts of pretreated cells, was identified as the interferon-inducible, double-stranded RNA (dsRNA)-regulated protein kinase (PKR). Depletion of ER Ca2+ stores did not alter the PKR contents of extracts. When incubated with reovirus dsRNA, extracts derived from cells with depleted ER Ca2+ stores displayed greater degrees of phosphorylation of PKR and of eIF-2 alpha than did control extracts. The enhanced dsRNA-dependent phosphorylation of PKR was observed regardless of prior induction of the kinase with interferon. Lower concentrations of dsRNA were required for maximal phosphorylation of PKR in extracts of treated as compared to control preparations. These findings suggest that PKR mediates the translational suppression occurring in response to perturbation of ER Ca2+ homeostasis.
Article
Hippocampal slices were successfully maintained for 24 hours in vitro in a flow-through chamber by using a modified artificial CSF (amino acids included). Measurement of energy metabolism parameters (adenine nucleotides) and the slice response to KCl-induced depolarization (release of GABA and aspartate) indicated that hippocampal slices were metabolically stable for at least 24 hours. The preparation was used to study recovery of protein synthesis after different periods of in vitro ischemia (5, 10, or 15 min). Protein synthesis inhibition was only partly reversed after 15 min of ischemia, but fully reversible after 5- or 10-min ischemia at 24 hours of recovery. Furthermore, the model was used to study a possible role of glutamate in postischemic inhibition of protein synthesis. Glutamate receptor agonists (glutamate or quinolinic acid) or antagonist (kynurenic acid) were applied during ischemia. Neither treatment affected the late (24 hours) outcome of ischemia, arguing against the critical role of glutamate in ischemic cell damage. The present approach allows use of the hippocampal slice preparation in the study of delayed effects of ischemia of different duration.
Article
Intra-ischemic moderate hypothermia generally protects the brain against ischemic cell death, while hypothermia instigated several hours into the reperfusion phase is considered to be less effective. Here we report the effect of hypothermia (32.5 degrees - 33.5 degrees C) of 5-h duration, initiated at 2, 6, 12, 24 and 36 h into the recirculation phase following 10 min of transient cerebral ischemia, on ischemic neuronal injury in the hippocampus and striatum of the rat. Hypothermia induced at 2 h, and 6 h postischemia reduces neuronal damage in the entire hippocampal CA1 region by approximately 50%. In the lateral CA1 region hypothermia induced at 12 h postischemia, significantly mitigates necrosis. When initiated at 2 h postischemia, but not later, protection was also observed in the striatum. Hypothermia induced 24 and 36 h postischemia was ineffective. A period of hypothermia of 5 h, initiated 2 h postischemia, was required for marked neuronal protection in the CA1 region, while 3.5-h hypothermia decreased neuronal damage by approximately 10% and 30 min hypothermia was ineffective. The clinical implications of the data are that extended period of hypothermia initiated long into the recovery phase following ischemia may prove beneficial. Hypothermia protects brain regions displaying rapid as well as delayed neuronal damage, and a minimal time of hypothermia is required for effective neuronal protection. Also, strict temperature control for up to 24 h postischemia may be required for proper assessment of the efficacy of cerebro-protective drugs.
Article
Hypothermia is a frequent occurrence in newborns, and thermoregulatory management is a fundamental part of medical stabilization. Although modest reduction in brain temperature (2-3 degrees C) before ischemia provides neuroprotection in adults, the effect of modest hypothermia on immature brain has not been examined. Nine-day-old swine were exposed to 15 min of incomplete global brain ischemia, with intraischemic rectal temperatures of either 38.3 +/- 0.4 degrees C (n = 10, normothermic) or 35.4 +/- 0.5 degrees C (n = 10, hypothermic). The relationship between rectal and brain temperature was delineated in preliminary experiments on four swine. Animals with intraischemic rectal temperatures maintained at either 39.5 degrees C or 35.5 degrees C were associated with a similar magnitude of difference in brain temperature. Therefore, rectal temperature was used to monitor brain temperature for 20 animals studied subsequently. Ischemia was induced by combining neck compression with hemorrhagic hypotension and resulted in similar group values for mean arterial pressure and changes in pH and blood gases at the completion of ischemia. A clinical overall performance score and brain tissue structure were evaluated after 72 h (or earlier if animals died prematurely). Hypothermic animals had less severe stages of impairment compared with the normothermic group (p = 0.023). Hypothermic piglets had less histologic damage in the neocortex at 0.5 cm beneath the brain surface (p = 0.048), the caudate nucleus (p = 0.038), and the pons/midbrain (p = 0.04) and the same direction of effect in neocortex at 1 cm beneath the surface (p = 0.07) and the cerebellum (p = 0.07) as compared with normothermic animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Recent studies in adult animals have shown that even small decreases in brain or core temperature ameliorate the damage resulting from hypoxic-ischemic insults. To determine the effect of minor reductions in ambient temperature either during or after an hypoxic-ischemic insult on the brain of the immature rat, 7-d-postnatal rat pups underwent unilateral common carotid artery ligation followed by exposure to hypoxia in 8% oxygen for 3 h. Control animals were maintained at 37 degrees C during hypoxia-ischemia. Intraischemic hypothermia was induced during the insult at temperatures of 34 degrees C and 31 degrees C. Postischemic hypothermia was induced by exposing rat pups that underwent hypoxia at 37 degrees C to recovering environments of 34 degrees C and 31 degrees C. Temperatures were recorded every 15 min from thermistor probes placed in the ipsilateral hemisphere and rectally. Neuropathologic alterations were assessed at 30 postnatal d. During hypoxia, animals became poikilothermic. Brain damage occurred in 90% of rat pups exposed to hypoxia-ischemia at 37 degrees C. Cerebral injury significantly decreased with decreasing temperatures during hypoxia-ischemia (p < 0.01). Only 30% of rats had brain damage when exposed to hypoxia-ischemia at 34 degrees C, and none of the rats exposed at 31 degrees C had brain damage. In contrast, there was no difference in the extent of cerebral injury between rat pups recovered under hypothermic conditions of either 34 degrees C or 31 degrees C compared with those recovered at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Regional protein synthesis of brain was measured by quantitative autoradiography in normo- and hypothermic rats submitted to 30 min of four-vessel occlusion. The tracer, [14C]leucine, was applied by controlled intravenous infusion to achieve constant plasma specific activity, and the admixture by proteolysis of unlabeled amino acids to the brain amino acid precursor pool was corrected by measuring the ratio of the labeled-to-unlabeled leucine distribution space in plasma and brain. In normothermic rats preischemic protein synthesis rate was 16.0 +/- 3.2, 9.2 +/- 3.4, 15.5 +/- 2.8, and 15.5 +/- 3.1 nmol of leucine/g/min (mean +/- SD) in the frontal cortex, striatum, hippocampal CA1 sector, and thalamus, respectively. After 30 min of ischemia at a constant brain temperature of 36 degrees C and a recirculation time of 1 h, protein synthesis was reduced in these regions to 6, 9, 8, and 36%, respectively. With ongoing recirculation, protein synthesis gradually returned to normal within 3 days in all areas except in the stratum pyramidale of the hippocampal CA1 sector where inhibition of neuronal protein synthesis was irreversible. Lowering of brain temperature to 30 degrees C during ischemia did not prevent the early global postischemic depression of protein synthesis, but promoted recovery to or above normal within 6 h in all areas including the stratum pyramidale of the CA1 sector. Improvement of protein synthesis in the CA1 sector was associated with improved neuronal survival, which increased from 1% in the normothermic to 69% in the hypothermic animals. These observations suggest that the protective effect of mild hypothermia on ischemic injury of the hippocampal CA1 sector is mediated by the reversal of the postischemic inhibition of protein synthesis.
Article
In fetal as well as newborn rats, acute hypoxic exposure results in significantly elevated brain ornithine decarboxylase (ODC) activity, polyamine concentrations, and ODC mRNA. The interpretations of these in vivo hypoxic-induced changes, however, are complicated by maternal confounding effects. To test the hypothesis that acute hypoxia will also increase ODC activity in vitro, we developed a brain slice preparation which eliminates such maternal effects. Sections of whole cerebrum, approximately 300-500 microns thick, were made from 3- to 4-day old Sprague-Dawley rat pups. The slices were equilibrated for 1 h in artificial cerebrospinal fluid (ACSF) continuously bubbled with 95% O2/5% CO2, prior to induction of hypoxia. We induced hypoxia by changing the oxygen concentration to 40%, 30%, 21%, 15%, 10%, or 0% O2, all with 5% CO2 and balance N2. In the normoxic control brain slices, low but stable basal ODC activity persisted for up to 5 h post-sacrifice. Slices in ACSF treated with bovine serum albumin (BSA), or both BSA and fetal bovine serum (FBS), however, showed stable ODC activity values 2- to 3-fold higher than slices in ACSF alone, for up to 5 h. In response to acute hypoxia (i.e., 15, 21, and 30% O2), ODC activity was elevated 1.5- to 2-fold above control values between 1 and 2 h after initiation of hypoxia. Qualitative light and electron microscopic examination of the neonatal brain slices following 2 h hypoxic exposure suggested that the great majority of cells did not show severe hypoxic damage or necrosis. It was concluded that: (1) in neonatal rat brain slices in vitro, stable ODC activity values approximating the whole brain ODC activity seen at sacrifice, can be maintained for several hours; (2) the in vivo hypoxic-induced increase in ODC activity can be approximated in vitro; (3) the neonatal rat brain slice preparation may be an alternative to other methods for studying hypoxic-induced ODC enzyme kinetics, or other brain enzymes, without maternal confounding effects; and (4) ODC activity may be an indicator of active metabolism within the newborn brain slice both in normoxia and hypoxia.
Article
Hypoxic-ischemic injuries can evolve over several days, and recent studies suggest that further neuronal death may occur 6 to 72 h later. Because cerebral temperature is an important determinant of outcome during the primary injury, we investigated the effect of temperature, on outcome, during the later phases of injury. Hypoxic-ischemic injury was induced in 21-d-old rats by unilateral ligation of the right carotid artery followed by exposure to 15 min of hypoxia of 8% O2 at 34 degrees C. Cerebral temperature changes were induced by modifying environmental temperature. The rats were divided into four treatment groups: group 1 (n = 15) remained at 34 degrees C for 72 h; group 2 (n = 14) were kept at 34 degrees C for 6 h and then at 22 degrees C for the remaining 66 h; group 3 (n = 17) remained at 22 degrees C for 6 h and 34 degrees C for the next 66 h; group 4 (n = 16) remained at 22 degrees C for 72 h. Rats kept at 22 or 34 degrees C had cortical temperatures of 35.5 +/- 0.1 degrees C and 37.9 +/- 0.2 degrees C, respectively. Histologic outcome was assessed 72 h after hypoxia. The area of cortical infarction was reduced in group 4 compared with groups 1-3 (p < or = 0.05). Striatal damage was reduced in group 4 (p = 0.05). Hippocampal neuronal loss was not significantly altered. In a subsequent study the area of cortical infarction was 12.1 +/- 3 mm2 in group 1 (n = 11) compared with 3.4 +/- 1.5 mm2 group 4 treated rats (n = 10) 21 d after the injury (p < 0.01). Thus hypothermia spanning both the first 6 h and from 6 to 72 h after injury was needed to improve outcome. Conversely exposure to the thermoneutral environment exacerbated the injury. These observations suggest that prolonged moderate cerebral hypothermia can be used to suppress the cytotoxic processes that occur after hypoxic-ischemic injury.
Article
We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors elF-4E, elF-4G, and elF-2 alpha to examine the effects of global brain ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal forebrain homogenates was approximately 0.4 fmol of leucine/min/microgram of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global brain ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of elF-4E. However, we observed in all 90-min-reperfused samples elF-4G fragments that also bound elF-4E. The amount of elF-2 alpha was not altered by ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated elF-2 alpha in normal samples or in those obtained after ischemia without reperfusion. However, serine-phosphorylated elF-2 alpha was uniformly present after 90 min of reperfusion and represented 24 +/- 3% of the elF-2 alpha in these samples. The serine phosphorylation of elF-2 alpha and partial fragmentation of elF-4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.
Article
It is widely accepted that disturbances of calcium homeostasis play a key role in the development of cell damage produced by transient cerebral ischemia. It is believed that the sharp increase in cytosolic calcium activity during ischemia activates a cascade of calcium-dependent metabolic processes which ultimately destroy the integrity of the cell. However, it has never been taken into account that ischemic cell damage may, at least in part, be caused by a disturbance of calcium homeostasis within the endoplasmic reticulum after transient cerebral ischemia. In fact, depletion of the endoplasmic reticulum from calcium induces metabolic changes resembling, in many respects, those produced by transient cerebral ischemia: it causes an inhibition of the activity of the eucaryotic initiation factor elF-2 alpha (by phosphorylation), a disaggregation of polyribosomes and thus an inhibition of global protein synthesis, and an increased expression of certain genes such as transcription factors (c-fos and c-jun) and the glucose-related protein grp78. Finally, a depletion of calcium in the endoplasmic reticulum induces tissue damage within the brain and triggers apoptosis in neuronal and non-neuronal cells. It is therefore concluded that cell damage induced by transient ischemia may, at least in part, be caused by a disturbance of calcium homeostasis within the endoplasmic reticulum.
Article
The influence of post-insult temperature modulation on ischemic injury in immature brain was studied in 7-day-old rats that underwent a unilateral carotid artery ligation followed by exposure to hypoxia in 8% oxygen at an ambient temperature of 36.5 degrees C. After the hypoxic exposure, the animals were separated into three groups and placed for 3 h in temperature-controlled incubators set at 32 degrees C, 35 degrees C, and 38 degrees C. In Study 1, the influence of post-insult temperature modulation was assessed after graded cerebral hypoxic-ischemic injury. Brain damage was assessed 1 week after the insult by comparison of wet weights in the cerebral hemispheres ipsilateral and contralateral to the carotid artery ligation. Rectal temperatures of the animals significantly correlated with extent of brain injury after 60 min (Spearman correlation coefficient, p = 0.44, P = 0.005) and 90 min (p = 0.46, P = 0.004) but not 120 min of hypoxia (p = 0.18, P = 0.46). In Study 2, animals were exposed to 75 min hypoxia, and injury was assessed morphometrically and histologically at 1 and 4 weeks after the injury. Rectal temperatures significantly correlated with the extent of ischemic injury in the cerebral cortex (p = 0.3, P = 0.046) and striatum (p = 0.3, P = 0.048) at 1 week, but not 4 weeks, after the insult. The findings indicate that post-insult hypothermia delayed the expression of mild to moderate brain damage by more than a week, after which the damage was as severe as in normothermic animals. The results indicate that the events that determine the final expression of a neonatal hypoxic-ischemic insult can be extended over a long interval by post-insult hypothermia.
Article
The use of hypothermia to mitigate cerebral ischemic injury is not new. From early studies, it has been clear that cooling is remarkably neuroprotective when applied during global or focal ischemia. In contrast, the value of postischemic cooling is typically viewed with skepticism because of early clinical difficulties and conflicting animal data. However, more recent rodent experiments have shown that a protracted reduction in temperature of only a few degrees Celsius can provide sustained behavioral and histological neuroprotection. Conversely, brief or very mild hypothermia may only delay neuronal damage. Accordingly, protracted hypothermia of 32–34°C may be beneficial following acute clinical stroke. A thorough mechanistic understanding of postischemic hypothermia would lead to a more selective and effective therapy. Unfortunately, few studies have investigated the mechanisms by which postischemic cooling conveys its beneficial effect. The purpose of this article is to evaluate critically the effects of postischemic temperature changes with a comparison to some current drug therapies. This article will stimulate new research into the mechanisms of lengthy postischemic hypothermia and its potential as a therapy for stroke patients.
Article
In developing brain, we have previously shown both in vivo [L.D. Longo, S. Packianathan, J.A. McQueary, R.B. Stagg, C.V. Byus and C.D. Cain, Acute hypoxia increases ornithine decarboxylase activity and polyamine concentrations in fetal rat brain, Proc. Natl. Acad. Sci. USA, Vol. 90 (1993) 692-696] and in vitro [S. Packianathan, C.D. Cain, B.H. Liwnicz and L.D. Longo, Ornithine decarboxylase activity in vitro in response to acute hypoxia: a novel use of newborn rat brain slices, Brain Res., Vol. 688 (1995) 61-71] that acute hypoxia is associated with a significant increase in ornithine decarboxylase (ODC) activity and polyamine concentrations. We tested the hypothesis that oxygen free radicals induce an increase in ODC activity similar to that of hypoxia and that both this and the hypoxia-induced response are inhibited by free radical scavengers. Slices of cerebrum, 300-500 microm thick, were made from P3 newborn Sprague-Dawley rat pups and equilibrated for 1 h in artificial cerebrospinal fluid continuously bubbled with 95% O2/5% CO2. Free radical-induced ODC activity response was measured beginning after a 1-h recovery period. Experiments were performed on slices treated with 5 X 10(-7) M xanthine (X) + 10 mU/ml xanthine oxidase (XO), with or without the free radical scavengers superoxide dismutase (SOD; 100 U/ml), catalase (CAT; 700 U/ml) or glutathione peroxidase (GPX; 3 U/ml). We also quantified slice malonaldehyde concentrations in response to hypoxia (21% O2/5% CO2/74% N2). Under control conditions, ODC activity was stable during the 2-h post-recovery period. In response to X/XO treatment, ODC activity increased 2.3-fold at 1.5 h post-recovery. In examining ODC activity as a function of xanthine dose, we noted that ODC activity increased in response to 2.5 X 10(-7) M xanthine; however, it decreased in response to 7.5 X 10(-7) M or higher concentrations. Free radical-induced ODC activity was significantly decreased by addition of the free radical scavengers, SOD, CAT or GPX. In addition, the hypoxic-induced increases in ODC activity and malonaldehyde concentration was also eliminated by the addition of SOD with CAT. (1) Oxygen free radicals, particularly hydroxyl radical (OH.), appear to trigger an induction of ODC activity in newborn rat cerebrum slices. (2) Oxygen free radicals also appear to mediate the hypoxic-induced increase in ODC activity. (3) Any consequent increase in polyamine synthesis may have profound effects on neurogenesis and neurodifferentiation in the developing brain.
Article
We have previously shown that mild hypothermia applied after hypoxia-ischemia in newborn piglets and rats reduces brain injury evaluated 3-7 d after the insult. The aim of the present study was to assess the neuroprotective efficacy of hypothermia with respect to short- (neuropathology) and long-term (neuropathology and sensorimotor function) outcome after hypoxia-ischemia in 7-d-old rats. One hundred fourteen animals from 13 litters survived either 1 or 6 wk after a hypoxic-ischemic insult. The animals were randomized to either 1) normothermic recovery for the whole 1- or 6-wk period or 2) cooling to a rectal temperature of 32.0 degrees C for the first 6 h followed by normothermic recovery with the dam. Hypothermia offered a uniform protection of 27, 35, 28, and 25% in cerebral cortex, hippocampus, basal ganglia, and thalamus, respectively, in the 1-wk survivors (n = 32). The corresponding values for the 6-wk survivors (n = 61) were 22, 28, 37, and 35%. There was a significant correlation between sensorimotor performance and infarct volume (r = 0.66; p < 0.001). However, the sensorimotor function was not significantly improved by hypothermia if all animals were included, but in female pups the total functional score was higher in the hypothermia group (150 +/- 35 versus 100 +/- 34, p < 0.0007) which corresponded to a marked (51%) reduction of the neuropathology score in this subgroup. This is the first neonatal study to show a long-term histopathologic protection of the brain after posthypoxic hypothermia.
Article
Cerebral hypothermia has been shown to reduce damage from experimental hy-poxia-ischemia if started shortly after reperfusion. However, in the newborn infant it may not be feasible to determine prognosis so soon after exposure to asphyxia. The aim of this study was to determine whether head cooling, delayed until shortly before the onset of postasphyxial seizure activity, is neuroprotective. Unanesthetized near-term fetal sheep in utero were subjected to 30 minutes of cerebral ischemia. Later, at 5.5 hours, they were randomized to either cooling (n = 7) or sham cooling (n = 10) for 72 hours. Intrauterine cooling was induced by circulating cold water through a coil around the fetal head. The water temperature was titrated to reduce fetal extradural temperature from 39.1 +/- 0.1 degreesC to between 30 degreesC and 33 degreesC, while maintaining esophageal temperature >37 degreesC. Cerebral cooling suppressed the secondary rise in cortical impedance (a measure of cytotoxic edema), but did not prevent delayed seizures, 8 to 30 hours after ischemia. Transient metabolic changes including increased plasma lactate and glucose levels were seen with a moderate sustained rise in blood pressure. This severe cerebral insult resulted in depressed residual parietal electroencephalographic activity after 5 days recovery (-14.2 +/- 1.5 decibels), associated with a watershed distribution of neuronal loss (eg, 94 +/- 4% in parasagittal cortex and 77 +/- 4% in the lateral cortex). Hypothermia was associated with better recovery of electroencephalographic activity (-8.9% +/- 1.8 decibels) and substantially reduced neuronal loss in the parasagittal cortex (46 +/- 13%), the lateral cortex (9 +/- 4%), and other regions except the cornu ammonis sectors 1 and 2 of the hippocampus. Delayed selective head cooling begun before the onset of postischemic seizures and continued for 3 days may have potential to significantly improve the outcome of moderate to severe hypoxic-ischemic encephalopathy.
Article
Considerable controversy exists about whether postischemic hypothermia can permanently salvage hippocampal CA1 neurons or just postpone injury. Studies of very brief cooling in rat have found transient benefit, whereas experiments in gerbil using protracted hypothermia report lasting protection. This discrepancy might be because of the greater efficacy of longer cooling or it might, for example, represent an important species difference. In the present study, a 48-hour period of mild hypothermia was induced starting 6 hours after 10 minutes of severe four-vessel occlusion ischemia in rats. Untreated normothermic ischemia resulted in total CA1 cell loss (99%), whereas delayed hypothermia treatment reduced neuronal loss to 14% at a 28-day survival. In unregulated rats, brain temperature spontaneously fell during ischemia, and stayed subnormal for an extended period after ischemia. This mild cooling resulted in more variable and less severe CA1 injury (75%). Finally, vertebral artery cauterization under halothane anesthesia caused an approximately 2 degrees C drop in brain temperature for 1 hour, but prevention of this hypothermia did not significantly affect CA1 damage. In summary, protracted postischemic hypothermia provided robust and long-term CA1 protection in rat. These results encourage the clinical assessment of prolonged hypothermia and its use as a model to understand ischemic CA1 injury.
Article
Prolonged cerebral hypothermia is neuroprotective if started within a few hours of hypoxia-ischemia. However, delayed seizure activity is one of the major clinical indicators of an adverse prognosis after perinatal asphyxia. The aim of this study was to determine whether head cooling delayed until after the onset of postasphyxial seizures may still be neuroprotective. Unanesthetized near-term fetal sheep in utero received 30 min of cerebral ischemia induced by bilateral carotid artery occlusion. Eight and one-half hours later, they received either cooling (n = 5) or sham cooling (n = 13) until 72 h after the insult. Intrauterine cooling, induced by circulating cold water through a coil around the fetal head, was titrated to reduce fetal extradural temperature from 39.4+/-0.1 degrees C to between 30 and 33 degrees C. Cerebral ischemia led to the delayed development of intense epileptiform activity from 6 to 8 h postinsult, followed by a marked secondary rise in cortical impedance (a measure of cytotoxic edema) and in carotid blood flow. Cerebral cooling markedly attenuated the secondary rise in impedance and reduced carotid blood flow (p < 0.001). After 5 d recovery, there was no significant difference in loss of parietal EEG activity relative to baseline in the hypothermia compared with the control group (-12.5+/-1.4 versus -15.2+/-1.2 dB, mean +/- SEM, NS) or in parasagittal cortical neuronal loss (82+/-9 versus 90+/-5%, NS). In conclusion, delayed prolonged head cooling begun after the onset of postischemic seizures was not neuroprotective. These data highlight the importance of intervention in the latent phase, after reperfusion but before the onset of secondary injury.
Article
Perinatal brain damage in the mature fetus is usually brought about by severe intrauterine asphyxia following an acute reduction of the uterine or umbilical circulation. The areas most heavily affected are the parasagittal region of the cerebral cortex and the basal ganglia. The fetus reacts to a severe lack of oxygen with activation of the sympathetic-adrenergic nervous system and a redistribution of cardiac output in favour of the central organs (brain, heart and adrenals). If the asphyxic insult persists, the fetus is unable to maintain circulatory centralisation, and the cardiac output and extent of cerebral perfusion fall. Owing to the acute reduction in oxygen supply, oxidative phosphorylation in the brain comes to a standstill. The Na(+)/K(+) pump at the cell membrane has no more energy to maintain the ionic gradients. In the absence of a membrane potential, large amounts of calcium ions flow through the voltage-dependent ion channel, down an extreme extra-/intracellular concentration gradient, into the cell. Current research suggests that the excessive increase in levels of intracellular calcium, so-called calcium overload, leads to cell damage through the activation of proteases, lipases and endonucleases. During ischemia, besides the influx of calcium ions into the cells via voltage-dependent calcium channels, more calcium enters the cells through glutamate-regulated ion channels. Glutamate, an excitatory neurotransmitter, is released from presynaptic vesicles during ischemia following anoxic cell depolarisation. The acute lack of cellular energy arising during ischemia induces almost complete inhibition of cerebral protein biosynthesis. Once the ischemic period is over, protein biosynthesis returns to pre-ischemic levels in non-vulnerable regions of the brain, while in more vulnerable areas it remains inhibited. The inhibition of protein synthesis, therefore, appears to be an early indicator of subsequent neuronal cell death. A second wave of neuronal cell damage occurs during the reperfusion phase. This cell damage is thought to be caused by the post-ischemic release of oxygen radicals, synthesis of nitric oxide (NO), inflammatory reactions and an imbalance between the excitatory and inhibitory neurotransmitter systems. Part of the secondary neuronal cell damage may be caused by induction of a kind of cellular suicide programme known as apoptosis. Knowledge of these pathophysiological mechanisms has enabled scientists to develop new therapeutic strategies with successful results in animal experiments. The potential of such therapies is discussed here, particularly the promising effects of i.v. administration of magnesium or post-ischemic induction of cerebral hypothermia.
Article
The effect of 24h of hypothermic recovery on moderate hypoxic-ischemic brain damage in P7-rats was investigated for 42d after the insult, using magnetic resonance and histopathology. Occlusion of right common carotid artery and 90min exposure to 8% O2 at 37°C body temperature produced cytotoxic edema of 51 (±11)% brain volume (BV) and depression of brain energy metabolism (PCr/Pi) from 1.43(±0.21) to 0.14(±0.11). During recovery, the body temperature was reduced to 30°C for 24h in 36 animals, but was kept at 37°C in 34 animals. The edema waned upon reoxygenation leaving only the core lesion at 2h, but reappeared reaching a maximal extent of 11 ±8% BV under hypothermia compared to 45(±10)% under normothermia at around 24h. PCr/Pi recovered transiently within 13h and declined again to 1.07(±0.19) under hypothermia and to 0.48(±0.22) under normothermia at around 24h. Hypothermia led to significant long term brain protection, leaving permanent tissue damage of 12(±6)% BV compared to 35(±12)% BV under normothermia. However, animals with severe initial injury developed large infarctions, despite hypothermic treatment. Even then, the time to develop infarction was significantly prolonged, leaving the opportunity for additional therapeutic intervention.
Postischemic hypothermia
  • F Colbourne
  • G Sutherland
  • D Corbett
Protein synthesis after global ischemia and selective vulnerability
  • Ka Hossmann
  • R Widmann
  • C Wiessner
  • E Dux
  • B Djuricic
  • G Röhn
Dramatic neuronal rescue with prolonged selective head cooling after ischemia in fetal lambs
  • Aj Gunn
  • Tr Gunn
  • Hh De Haan
  • Ce Williams
  • Pd Gluckman
Hibernation and marmot physiology. Publication no. 494
  • Fc Benedikt
  • Rc Lee
  • Bigelow WG