Increased survival of embryonic nigral neurons when grafted to hypothermic rats.

Hypothermia can reduce neuronal death caused by ischemia and traumatic brain injury. We therefore investigated whether mild hypothermia in rats receiving a transplant of embryonic mesencephalic rat tissue increases survival of the implanted dopaminergic neurons. Mild hypothermia (32-33 degrees C) during graft implantation and for the following 90 min significantly increased the survival of transplanted dopaminergic neurons to 171% of control values in normothermic (37 degrees C) rats. This demonstrates that treatment of the graft recipient for a relatively short period during and after surgery has a favorable effect on the survival of grafted dopaminergic neurons. These findings may be of importance for clinical neural transplantation trials which are in need of procedures that improve transplant survival.

The effect of alpha-phenyl-tert-butyl nitrone (PBN) on free radical formation in transient focal ischaemia measured by microdialysis and 3,4-dihydroxybenzoate formation.

alpha-phenyl-tert-butyl nitrone (PBN) reduces infarct size, improves recovery of brain energy metabolism and delays the secondary increase in extracellular potassium after focal ischaemia, presumably by trapping OH radicals. We investigated the effect of PBN on the formation of 3,4-dihydroxybenzoic acid (3,4-DHBA) as a measure of OH radical formation, during and following middle cerebral artery occlusion (MCAO). Rats, subjected to 2 h of ischaemia followed by 3 h of recirculation, were injected with either vehicle or PBN (100 mg kg-1 i.p.) prior to MCAO or immediately after recirculation, respectively. The in vivo microdialysis technique was used to collect samples for analysis of 3,4-DHBA by HPLC. The basal levels of 3,4-DHBA were 56-77 nmol L-1 in the four groups. During ischaemia, the formation of 3,4-DHBA decreased by about 50% in all groups. Upon recirculation, a 3-fold rise in 3,4-DHBA formation was seen. At 2 h of recirculation the mean value of 3,4-DHBA in the pretreated, vehicle-injected animals was 125 +/- 18 nmol L-1 and in the PBN-injected 145 +/- 48 nmol L-1, respectively. When the animals were treated after MCAO either with vehicle or PBN the values at 2 h recirculation were 155 +/- 148 and 189 +/- 145 nmol L-1, respectively. No statistically significant difference between vehicle- and PBN-injected groups was seen. We conclude that during reperfusion following MCAO, hydroxyl radical formation increases. The increase is not ameliorated by PBN which suggests that PBN does not protect the brain by a general scavenging of OH radicals, although tissue specific actions cannot be excluded.

Calcium metabolism of focal and penumbral tissues in rats subjected to transient middle cerebral artery occlusion.

The present experiments were undertaken to define changes in tissue calcium metabolism in focal and perifocal ("penumbral") tissues following 2 h of transient middle cerebral artery occlusion (MCAO) in rats, induced with an intraluminal filament occlusion technique. The extracellular calcium concentration ([Ca2+]e) was measured with ion-selective microelectrodes in neocortical focus and penumbra. For measurement of total tissue calcium content, tissue samples from these areas were collected and analyzed with atomic absorption spectrometry. During MCAO, [Ca2+]e in a neocortical focal area fell from a normal value of about 1.2 mM to values around 0.1 mM, suggesting translocation of virtually all extracellular calcium to intracellular fluids. Recirculation was accompanied by re-extrusion of calcium within 5-7 min; however, [Ca2+]e never returned to normal but stabilized at about 50% of the control value for the first 6 h, and decreased further after 24 h. In penumbral areas, [Ca2+]e showed the expected transient decreases associated with spreading depression-like (or ischemic) depolarization waves. Recirculation was followed by return of [Ca2+]e towards normal values. In the focus, water content increased from about 79% to about 80.4% at the end of the 2-h period of ischemia. After 2 h and 4 h of recirculation, the edema was aggravated (mean values 81.9% and 81.2%, respectively). After 6 h and 24 h, the edema was more pronounced (83.6% and 83.8%, respectively). In the penumbra, no significant edema was observed until 6 h and 24 h of recirculation. The total tissue calcium content in the focus (expressed by unit dry weight) increased at the end of the ischemia period demonstrating calcium translocation from blood to tissue. After 6 h and 24 h, the content increased two- to threefold, compared with control. Changes in the penumbra were qualitatively similar but less pronounced, and a significant increase was not observed until after 6 h of recirculation. The results suggest that 2 h of MCAO leads to a profound perturbation of cell calcium metabolism. In focal areas, cells fail to extrude the calcium that is gradually accumulated during reperfusion and show massive calcium overload after the first 4-6 h of recirculation. Penumbral tissues show a similar increase in calcium concentration after 6 h of recirculation.

Extracellular potassium in a neocortical core area after transient focal ischemia.

BACKGROUND AND PURPOSE: Occlusion of the middle cerebral artery (MCAO) results in bioenergetic failure in the densely ischemic core areas. During reperfusion, transient recovery of the bioenergetic state is followed by secondary deterioration. In this study, we recorded the extracellular potassium concentrations in the cortical core during 2 hours of MCAO, as well as during recovery. One group of animals with recirculation periods of 6 to 8 hours was given the free radical spin trap alpha-phenyl-N-tert-butyl nitrone (PBN). METHODS: The experiments were performed on adult male Wistar rats (305 to 335 g). The right MCA was occluded by an intraluminal filament technique. For [K+]e measurements a craniotomy was made over the right cortex, and an ion-sensitive microelectrode was lowered into the ischemic focus. Recording of [K+]e was continued for 2 hours. After 48 hours of reperfusion, infarction size was estimated with 2,3,5-triphenyltetrazolium chloride. RESULTS: During MCA occlusion, [K+]e rose to approximately 60 mmol/L. However, several animals showed transient (and partial) periods of repolarization accompanied by a decrease in [K+]e. Immediately on reperfusion, the [K+]e started to recover and reached baseline levels (2.5 mmol/L) within 3 to 5 minutes. During the first 6 hours of recovery, [K+]e was stable at about 2.5 mmol/L, but after this period a moderate increase in the [K+]e was observed. This was not observed in animals injected with PBN 1 hour after reperfusion. CONCLUSIONS: The data suggest that delayed cell membrane dysfunction, as reflected in a rise in [K+]e, occurs after about 6 hours of reperfusion and that treatment with PBN in a single dose ameliorates or delays such deterioration of plasma membrane function.

The influence of acidosis on hypoglycemic brain damage.

The objective of the study was to explore whether hypoglycemic brain damage is affected by super-imposed acidosis. To that end, animals with insulin-induced hypoglycemic coma, defined in terms of a negative DC potential shift, massive release of K+, or cellular uptake of Ca2+, were exposed to excessive hypercapnia (PaCO2 approximately 200 or approximately 300 mm Hg) during the last 25 min of the 30-min coma period. Animals were allowed to survive for 7 days before their brains were fixed by perfusion, and the cell damage was assessed by light microscopy. Other animals were analyzed with respect to changes in extracellular pH (pHe) or extracellular K+ or Ca2+ concentrations (K+e and Ca2+e, respectively). The total CO2 content (TCO2) was also measured to allow derivation of intracellular pH (pHi). The increase in PaCO2 to 190 +/- 15 and 312 +/- 23 mm Hg (means +/- SD) reduced the pHe from a predepolarization value of approximately 7.4 and a postdepolarization value (after the first 5 min of coma) of approximately 7.3 to 6.8 and 6.7, respectively. The corresponding mean pHi values were 6.7 and 6.5. The hypercapnia did not alter the K+e, which rose to 50-60 mM at the onset of hypoglycemic coma, but it increased the Ca2+e from approximately 0.05 to 0.10-0.16 mM. Normocapnic animals with induced hypoglycemic coma of 30-min duration showed the expected neuronal lesions in the neocortex, hippocampus, and caudoputamen. Hypercapnia clearly aggravated this damage, particularly in the caudoputamen, subiculum, and CA1 region of the hippocampus, and caused additional damage to cells in the CA3 region and piriform cortex. A rise in CO2 tension from approximately 200 to 300 mm Hg did not further aggravate the damage. The results thus demonstrate that relative moderate acidosis aggravates damage that is believed to be mostly neuronal, sparing glia cells and vascular tissue.

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.

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.

Functional, metabolic, and circulatory changes associated with seizure activity in the postischemic brain.

The present study was undertaken to explore how transient ischemia in rats alters cerebral metabolic capacity and how postischemic metabolism and blood flow are coupled during intense activation. After 6 h of recovery following transient forebrain ischemia 15 min in duration, bicuculline seizures were induced, and brains were frozen in situ after 0.5 or 5 min of seizure discharge. At these times, levels of labile tissue metabolites were measured, whereas the cerebral metabolic rate for oxygen (CMRO2) and cerebral blood flow (CBF) were measured after 5 min of seizure activity. After 6 h of recovery, and before seizures, animals had a 40-50% reduction in CMRO2 and CBF. However, because CMRO2 rose three-fold and CBF fivefold during seizures, CMRO2 and CBF during seizures were similar in control and postischemic rats. Changes in labile metabolites due to the preceding ischemia encompassed an increased phosphocreatine/creatine ratio, as well as raised glucose and glycogen concentrations. Seizures gave rise to minimal metabolic perturbation, essentially comprising reduced glucose and glycogen contents and raised lactate concentrations. It is concluded that although transient ischemia leads to metabolic depression and a fall in CBF, the metabolic capacity of the tissue is retained, and drug-induced seizures lead to a coupled rise in metabolic rate and blood flow.

The influence of repeated spreading depression-induced calcium transients on neuronal viability in moderately hypoglycemic rats.

The calcium transients which are associated with spreading depression (SD) do not lead to neuronal necrosis, even if the SDs are repeated over hours. We have previously shown that a restriction of energy production by moderate hypoglycemia prolongs the calcium transients during SD. In the present experiments, we explored whether such prolonged transients lead to neuronal necrosis. To that end, SDs were elicited for 2 h by topical application of KCl in anesthetized rats at plasma glucose concentrations of 6, 3, and 2 mM. The animals were then allowed to recover, and they were studied histopathologically after 7 days. In two other groups, hypoglycemic coma of 5 min duration (defined in terms of the d.c. potential shift) was induced either without or with a preceding train of SDs. These animals were also evaluated with respect to histopathological alterations. SDs elicited for 2 h did not give rise to neuronal damage when elicited at plasma glucose concentration of 6 mM, and, of the animals maintained at 3 and 2 mM, only a few animals showed (mild) damage. In general, therefore, repeated SDs with calcium transients of normal or increased duration fail to induce neuronal damage. The results suggest that, if calcium transients are responsible for a gradual extension of the infarct into the penumbra zone of a focal ischemic lesion some additional pathophysiological factors must be present, such as overt energy failure, acidosis, or microvascular damage. A hypoglycemia-induced calcium transient of 5 min duration gave no or only moderate neuronal damage. However, if a series of SDS were elicited in the precoma period, the damage was exaggerated.(ABSTRACT TRUNCATED AT 250 WORDS)

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 approximately 1.2 to approximately 0.02 mM, i.e., to approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid increase of BDNF mRNA levels in cortical neurons following spreading depression: regulation by glutamatergic mechanisms independent of seizure activity.

Levels of mRNA for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and the tyrosine kinase receptors trkB and trkC have been studied using in situ hybridization in the rat brain after topical application of KCl to the cortical surface (which induces spreading depression). Repeated episodes of spreading depression during 2 h caused a rapid and marked increase of BDNF mRNA levels in deep and, in particular, superficial cortical layers of the ipsilateral hemisphere (to 213 and 417% of control, respectively). Maximal levels were reached within 2 h after the cessation of spreading depression and at 24 h BDNF mRNA expression had returned to control values. Levels of BDNF mRNA were unaffected in the hippocampus, in areas outside the cerebral cortex and in the contralateral hemisphere. Furthermore, no change of the expression of mRNA for NGF, NT-3, trkC or the full length trkB receptor was detected at any time point. However, at 2 h after spreading depression there was an increased level (150% of control) in superficial cortical layers of mRNA hybridizing to an oligonucleotide probe detecting both truncated receptors lacking the tyrosine kinase domain and full length trkB receptors. Also one single episode of spreading depression gave rise to a significant increase of cortical BDNF mRNA levels (to 207% of control), which was attenuated (by 61%) after administration of the competitive NMDA receptor antagonist CGS 19755. The results provide evidence that mild brain insults associated with glutamate release and elevated intracellular calcium, such as spreading depression, also in the absence of seizure activity can lead to activation of the BDNF gene in cortical neurons.

Recovery of mitochondrial and plasma membrane function following hypoglycemic coma: coupling of ATP synthesis, K+ transport, and changes in extra- and intracellular pH.

The primary objective of the present study was to evaluate the recovery of plasma and mitochondrial membrane functions after 30 min of hypoglycemic coma and to establish whether a lingering accumulation of free fatty acids (FFAs) delays the recovery. A secondary objective was to study whether production of metabolic acids following glucose infusion leads to a fall in intracellular pH (pHi). Phosphocreatine, creatine, ATP, ADP, and AMP, as well as glycogen, glucose, lactate, pyruvate, and FFAs of rat brain cortex and caudoputamen were measured, and "free" ADP was calculated from the creatine kinase equilibrium. Extracellular pH (pHe) and K+ concentration (K+e) were measured with ion-sensitive microelectrodes, and pHi was derived by the CO2 method. Glucose injection was followed by resumption of oxidative phosphorylation within approximately 2 min and by an equally rapid restoration of normal K+e levels. These functions recovered although tissue FFAs remained elevated for at least 7-8 min. Tissue lactate content increased only moderately and production of metabolic acids did not lead to intracellular acidosis. After 15 min of recovery, pHi was moderately increased, although pHe fell toward 7.0. It is speculated that the dissociation between intra- and extra-cellular pH is compatible with an up-regulation of an Na+/H+ antiporter, e.g., by phosphorylation.

Influence of plasma glucose concentration on rat brain extracellular calcium transients during spreading depression.

The objective of this study was to establish whether tissues that are energy compromised, but not energy depleted, demonstrate exaggerated calcium transients when subjected to membrane depolarizations of the spreading depression (SD) type. Anesthetized and artificially ventilated rats were given insulin in order to induce progressively lower plasma glucose concentrations. Spreading depression was elicited by local application of KCl; extracellular calcium concentration (Ca2+e) as well as direct current (DC) potential were recorded. When plasma glucose concentration fell below approximately 3 mM, the duration of the Ca2+e transient gradually increased to values exceeding 500% of control. The increase was associated with a corresponding increase in the duration of the DC potential shift, but the amplitude of the Ca2+e transient did not change. It is concluded that a restriction of glucose (or oxygen) supply, as occurs in hypoglycemia (or hypoxia), prolongs the calcium transient associated with depolarization of the SD type, even though tissue phosphocreatinine and ATP concentrations are normal. The results support the contention that repeated depolarizations, occurring in the penumbral zone of a focal ischemic lesion, could lead to calcium-related damage.

Influence of ischemia on blood-brain and blood-CSF calcium transport.

The objective was to explore whether the increased incorporation of 45Ca into selectively vulnerable neurons, observed after transient ischemia, can be explained by enhanced blood-tissue and/or enhanced blood-CSF transfer. Anesthetized rats were subjected to 15 min of forebrain ischemia, with recirculation for 20 or 60 min, or 24 h. The transfer constants (Kin) and unidirectional fluxes (Jin) for calcium in tissue and CSF were determined following i.v. injection of 45Ca, integration of the curve for plasma-specific activity over 10 min, and sampling of cisternal CSF, and tissue (cortex, caudoputamen, hippocampus, and cerebellum). Predictably, values for Kin and Jin in control animals were much larger in CSF than in tissues, and hippocampus had higher values than the other areas, probably because of its closeness to the lateral ventricle. Ischemia failed to alter the Kin and Jin values, demonstrating that the low permeability of blood-brain and blood-CSF barriers to calcium is upheld both in the immediate recirculation period, and after 24 h of recirculation. The results support the contention that the increased 45Ca incorporation during recovery is due to increased calcium cycling across functionally altered cell membranes.