Association of the bleeding time test with aspects of traumatic brain injury in patients with alcohol use disorder.

BACKGROUND-AIM: Traumatic brain injury (TBI) and alcohol use disorder (AUD) can occur concomitantly and be associated with coagulopathy that influences TBI outcome. The use of bleeding time tests in TBI management is controversial. We hypothesized that in TBI patients with AUD, a prolonged bleeding time is associated with more severe injury and poor outcome. MATERIAL AND METHODS: Moderate and severe TBI patients with evidence of AUD were examined with bleeding time according to IVY bleeding time on admission during neurointensive care. Baseline clinical and radiological characteristics were recorded. A standardized IVY bleeding time test was determined by staff trained in the procedure. Bleeding time test results were divided into normal (≤ 600 s), prolonged (> 600 s), and markedly prolonged (≥ 900 s). Normal platelet count (PLT) was defined as > 150,000/µL. This cohort was compared with another group of TBI patients without evidence of AUD. RESULTS: Fifty-two patients with TBI and AUD were identified, and 121 TBI patients without any history of AUD were used as controls. PLT was low in 44.2% and bleeding time was prolonged in 69.2% of patients. Bleeding time values negatively correlated with PLT (p < 0.05). TBI patients with markedly prolonged values (≥ 900 s) had significantly increased hematoma size, and more frequently required intracranial pressure measurement and mechanical ventilation compared with those with bleeding times < 900 s (p < 0.05). Most patients (88%) with low platelet count had prolonged bleeding time. No difference in 6-month outcome between the bleeding time groups was observed (p > 0.05). Subjects with TBI and no evidence for AUD had lower bleeding time values and higher platelet count compared with those with TBI and history of AUD (p < 0.05). CONCLUSIONS: Although differences in the bleeding time values between TBI cohorts exist and prolonged values may be seen even in patients with normal platelet count, the bleeding test is a marker of primary hemostasis and platelet function with low specificity. However, it may provide an additional assessment in the interpretation of the overall status of TBI patients with AUD. Therefore, the bleeding time test should only be used in combination with the patient's bleeding history and careful assessment of other hematologic parameters.

Grafted neural progenitors migrate and form neurons after experimental traumatic brain injury.

PURPOSE: Neural stem and progenitor cells (NSPC) generate neurons and glia, a feature that makes them attractive for cell replacement therapies. However, efforts to transplant neural progenitors in animal models of brain injury typically result in high cell mortality and poor neuronal differentiation. METHODS: In an attempt to improve the outcome for grafted NSPC after controlled cortical impact we transplanted Enhanced Green Fluorescent Protein (EGFP)-positive NSPC into the contra lateral ventricle of mice one week after injury. RESULTS: Grafted EGFP-NSPC readily migrated to the injured hemisphere where we analyzed the proportion of progenitors and differentiated progeny at different time points. Transplantation directly into the injured parenchyma, resulted in few brains with detectable EGFP-NSPC. On the contrary, in more than 90% of the mice that received a transplant into the lateral ventricle detectable EGFP-positive cells were found. The cells were integrated into the lateral ventricle wall of the un-injured hemisphere, throughout the corpus callosum, and in the cortical perilesional area. At one-week post transplantation, grafted cells that had migrated to the perilesion area mainly expressed markers of neural progenitors and neurons, while in the corpus callosum and the ventricular lining, grafted cells with a glial fate were more abundant. After 3 months, grafted cells in the perilesion area were less abundant whereas cells that had migrated to the walls of the third- and lateral- ventricle of the injured hemisphere were still detectable, suggesting that the injury site remained a hostile environment. CONCLUSION: Transplantation to the lateral ventricle, presumably for being a neurogenic region, provides a favorable environment improving the outcome for grafted NSPC both in term of their appearance at the cortical site of injury, and their acquisition of neural markers.

Intracranial pressure changes during fluid percussion, controlled cortical impact and weight drop injury in rats.

BACKGROUND: In traumatic brain injury research, the fluid percussion injury (FPI) model in the rat is widely used. The injury is graded based on indirect criteria, such as the extracranial pressure wave and/or physiological responses to the injury. We designed this study to investigate if the extracranially monitored pressure in the FPI-device corresponded to the actual intracranial situation. Severe controlled cortical impact (CCI) and severe weight drop injury (WDI) were studied for comparison. METHOD: We tested the correlation between the extra- and intracranial pressures during severe FPI in rat (2.6-2.9 atm), using pressure probes (diameter 0.34 mm) with high frequency (500 Hz) and high pressure range (1-5 atm). The probes were inserted into either of the lateral ventricles in FPI and in the contralateral lateral ventricle in CCI and WDI to compare the ictal pressure pulses between the models. FINDINGS: FPI showed a time lag between the extracranial, intracranial ipsilateral and intracranial contralateral pressure curves respectively, reflecting the different distances between the pressure source and the individual pressure probes. There was a high degree of correlation (r = 0.994, p<0.0001) between the extra- and intracranial pressure pulses, once corrected for the time lag. We found no significant differences between the extracranial and the intracranial peak pressure in either ventricle in FPI. In CCI and WDI the contralateral pressure pulses were significantly smaller than in FPI. CCI resulted in higher pressure peaks than WDI, due to higher impact velocity. CONCLUSIONS: The extracranial pressure pulse appears to be a good estimate of the intraventricular pressure pulse generated during FPI. Severe CCI and WDI generated intraventricular pressure pulses of much lower magnitude than FPI, explaining the lesser degree of brain stem involvement in the former models.

Middle cerebral artery occlusion and reperfusion in primates monitored by microdialysis and sequential positron emission tomography.

BACKGROUND AND PURPOSE: In a previous investigation concerning the hemodynamic and metabolic changes over time displayed by sequential positron emission tomography (PET) in a middle cerebral artery (MCA) occlusion/reperfusion primate model, a metabolic threshold for irreversible ischemia could be identified (reduction of metabolic rate of oxygen [CMRO(2)] to approximately 60% of the contralateral hemisphere). To evaluate the potential of microdialysis (MD) as an instrument for chemical brain monitoring, the aim of this subsequent study was to relate the chemical changes in MD levels directly to the regional metabolic status (CMRO(2) above or below the metabolic threshold) and the occurrence of reperfusion, as assessed by PET. METHODS: Continuous MD (2 probes in each brain) and sequential PET measurements were performed during MCA occlusion (2 hours) and 18 hours (mean) of reperfusion in 8 monkeys (Macaca mulatta). Energy-related metabolites (lactate, pyruvate, and hypoxanthine) and glutamate were analyzed. The MD probe regions were divided into 3 categories on the basis of whether CMRO(2) was below or above 60% of the contralateral region (metabolic threshold level) during MCA occlusion and whether reperfusion was obtained: severe ischemia with reperfusion (n=4), severe ischemia without reperfusion (n=4), and penumbra with reperfusion (n=5). RESULTS: The lactate/pyruvate ratio, hypoxanthine, and glutamate showed similar patterns. MD probe regions with severe ischemia and reperfusion and probe regions with severe ischemia and no reperfusion displayed high and broad peaks, respectively, during MCA occlusion, and the levels almost never decreased to baseline. Penumbra MD probe regions displayed only slight transient increases during MCA occlusion and returned to baseline. CONCLUSIONS: This experimental study of focal ischemia showed that the extracellular changes of energy-related metabolites and glutamate differed depending on the ischemic state of the brain during MCA occlusion and depending on whether reperfusion occurred. If MD proves to be beneficial in clinical practice, it appears important to observe relative changes over time.

Respiratory activity of isolated rat brain mitochondria following in vitro exposure to oxygen radicals.

Respiratory activity of isolated rat brain mitochondria was measured following in vitro exposure to oxygen radicals. The radicals were generated by hypoxanthine and xanthine oxidase in the presence of a suitable iron chelate and caused a severe inhibition of respiration stimulated by phosphate plus ADP (with malate + glutamate as substrate). The damage could be prevented by catalase or high concentrations of mannitol, but not by superoxide dismutase. A similar effect was observed when hypoxanthine and xanthine oxidase were replaced by glucose and glucose oxidase or by hydrogen peroxide. Most of the findings indicate that the hydroxyl radical is the damaging agent. It is concluded that brain mitochondria exposed to oxygen radicals in vitro show an inhibition of respiratory activity similar to that reported by other investigators as occurring in mitochondria in vivo following transient cerebral ischemia. Therefore, oxygen radicals may contribute to this type of cell damage.

Influence of in vitro lactic acidosis and hypercapnia on respiratory activity of isolated rat brain mitochondria.

Respiratory activity and the ADP/O ratio of isolated rat brain mitochondria were measured following incubation with varying concentrations of lactic acid in reaction media buffered either with bicarbonate and CO2 or with phosphate alone, at a pH of 7.1. Increasing lactic acid levels caused a progressive decrease in substrate-, phosphate-, and ADP-stimulated (State 3) respiration and ADP/O ratios. Fifteen millimolar lactic acid, pH 6.4, caused approximately 50% inhibition of State 3 respiration (with malate + glutamate as substrate). At lower pH values (5.3-6.1), addition of ADP caused little or no increase in O2 consumption; i.e., ATP formation ceased. Addition of lactic acid at constant pH moderately affected respiratory control ratios but did not change State 3 respiration or ADP/O ratios. Thus, the effect of lactic acid was related to the pH change. Increasing CO2 concentrations in the reaction medium had similar effects on mitochondrial respiration, indicating that changes in extramitochondrial pH rather than in transmembrane H+ gradients determined the respiratory alterations. Following a 5-min incubation of mitochondria with lactic acid, pH 6.1, there was an incomplete recovery of State 3 respiration and respiratory control ratios. It is concluded that mitochondrial respiration is inhibited by a decrease in pH which, if excessive, may lead to a permanent suppression of ATP production. These results may, at least partly, explain the deleterious effects of enhanced lactic acidosis in brain ischemia.

Mitochondrial response to transient forebrain ischemia and recirculation in the rat.

Recovery of brain mitochondrial function was studied following forebrain ischemia induced in rats by common carotid artery occlusion in combination with hypotension caused by bleeding. A reversible insult was induced by 15-min ischemia in fasted animals (hypoglycemic ischemia), and an irreversible one by 30-min ischemia in fed animals (normoglycemic ischemia), the latter procedure causing exaggerated lactic acidosis as well. Mitochondrial function recovered during a 30-min recirculation period after 15-min hypoglycemic ischemia, although a small amount of Ca2+ accumulated during recirculation. Thirty-minute normoglycemic ischemia induced irreversible mitochondrial damage that was not associated with Ca2+ accumulation during recirculation. Ischemia of 15 and 30 min caused a loss of mitochondrial Mg2+ (approximately 25%) that persisted during recirculation but did not influence recovery. Based on our earlier data obtained on isolated brain mitochondria in vitro, it is suggested that the lack of full recovery following 30 min of normoglycemic ischemia was due to the profound lactic acidosis during this insult.

Changes in cortical extracellular levels of energy-related metabolites and amino acids following concussive brain injury in rats.

The aim of this study was to measure extracellular chemical changes in the cerebral cortex in response to compression contusion trauma in rats. Energy-related metabolites (i.e., lactate, pyruvate, adenosine, inosine, and hypoxanthine) and amino acids were harvested from the extracellular fluid (ECF) using microdialysis and analyzed by high-performance liquid chromatography. The measurements were performed in cortical tissue, where neuronal injury occurs in this model. The severity of the trauma was varied by using different depths of impact: mild trauma, 1.5 mm; severe trauma, 2.5 mm. The trauma induced a dramatic increase in the ECF levels of energy-related metabolites that was conditioned by the severity of the insult. The ECF level of taurine, glutamate, aspartate, and gamma-aminobutyric acid (GABA) also rose markedly, while other amino acids did not change significantly. The results suggest that the trauma induced a transient, profound focal disturbance of energy metabolism in the cortical tissue, probably as a result of mechanically induced disruption of ion homeostasis and reduced blood flow in combination. The data support the potential role of glutamate and aspartate as mediators of traumatic brain injury. However, the concomitantly released adenosine, GABA, and taurine may be protective and ameliorate excitotoxicity. In analogy with the reported cumulative damaging effects of repeated ischemic insults, the observed ECF changes may help explain the vulnerability of traumatized brain tissue to secondary ischemia.

Lactic acidosis and recovery of mitochondrial function following forebrain ischemia in the rat.

The effect of different degrees of lactic acidosis on the recovery of brain mitochondrial function, measured as respiratory activity in isolated mitochondria or cortical concentrations of labile phosphates and carbohydrate substrates, was studied during 30 min of recirculation following 15 min of near-complete forebrain ischemia in rats. During ischemia, there was a marked decrease in mitochondrial State 3 respiration in vitro and a depletion of energy stores (i.e., phosphocreatine, ATP, glucose, and glycogen) in vivo that was similar in the high- and low-lactate ischemia groups. However, lactate concentrations differed markedly (20 and 10 mumol g-1, respectively). During recirculation, there was a near-complete recovery of both respiratory activity in vitro and adenylate energy charge (EC) in vivo regardless of the differences in lactic acidosis during ischemia. Respiratory activity and EC were well correlated. The changes in Ca2+ homeostasis during ischemia, an increase in tissue and a decrease in mitochondrial Ca2+ content, were reversed rapidly after ischemia in both high- and low-lactate ischemia animals and did not hinder an early recovery of mitochondrial function. It is concluded that lactic acidosis, with lactate levels reaching 20 mumol g-1 during 15-min ischemia, does not adversely affect early postischemic recovery of mitochondrial function.

Effects of the nitrone radical scavengers PBN and S-PBN on in vivo trapping of reactive oxygen species after traumatic brain injury in rats.

In previous studies, the authors showed that the nitrone radical scavenger alpha-phenyl-N- tert -butyl nitrone (PBN) and its sulfo-derivative, 2-sulfo-phenyl-N- tert -butyl nitrone (S-PBN), attenuated cognitive disturbance and reduced tissue damage after traumatic brain injury (TBI) in rats. In the current study, the production of reactive oxygen species (ROS) after TBI was monitored with microdialysis and the 4-hydroxybenzoic acid (4-HBA) trapping method. A single dose of PBN (30 mg/kg) or an equimolar dose of S-PBN (47 mg/kg) was administered intravenously 30 minutes before a controlled cortical contusion injury in rats. Plasma and brain tissue drug concentrations were analyzed at the end of the microdialysis experiment (3 hours after injury) and, in a separate experiment with S-PBN, at 30 and 60 minutes after injury. Traumatic brain injury caused a significant increase in ROS formation that lasted for 60 minutes after the injury as evidenced by increased 3,4-dihydroxybenzoic acid (3,4-DHBA) concentrations in the dialysate. PBN and S-PBN equally and significantly attenuated the posttraumatic increase in 3,4-DHBA formation. High PBN concentrations were found bilaterally in brain tissue up to 3 hours after injury. In contrast, S-PBN was rapidly cleared from the circulation and was not detectable in brain at 30 minutes after injury or at any later time point. The results suggest that scavenging of ROS after TBI may contribute to the neuroprotective properties observed with nitrone spin-trapping agents. S-PBN, which remained undetectable even in traumatized brain tissue, reduced ROS production to the same extent as PBN that readily crossed the blood-brain barrier. This finding supports an important role for ROS production at the blood-endothelial interface in TBI.

Dynamics of extracellular metabolites in the striatum after middle cerebral artery occlusion in the rat monitored by intracerebral microdialysis.

The aim of this study was to measure changes in the extracellular fluid (ECF) concentration of lactate, pyruvate, purines, amino acids, dopamine, and dopamine metabolites in the striatum of rats subjected to focal cerebral ischemia, using intracerebral microdialysis as the sampling technique. Microdialysis probes were inserted into the lateral part of the caudate-putamen bilaterally 2 h before the experiment. Ischemia was induced by permanent middle cerebral artery occlusion (MCAO) on the left side. Microdialysis samples were analyzed by high performance liquid chromatography. Following MCAO, the concentration of lactate, adenosine, inosine, and hypoxanthine rose markedly in the ECF on the occluded side, while there was no significant change in pyruvate. These changes were accompanied by dramatically elevated levels of aspartate, glutamate, taurine, gamma-aminobutyric acid, and dopamine. There was also a marked increase in alanine/tyrosine, while minor or no changes occurred with other amino acids. Concomitantly, the ECF level of the dopamine metabolites 3,4-dihydroxyphenylacetate and homovanillic acid decreased. There was no significant increase in any of the metabolites measured on the right, nonoccluded side. In relation to the concept of excitotoxicity in brain ischemia, it is concluded that during the acute stage of focal cerebral ischemia, the ECF is flooded with both potentially harmful (e.g., aspartate, glutamate, and DA) and protective (e.g., taurine, GABA, and adenosine) agents. The relative importance of these events for the development of cell death in the ischemic penumbra needs to be elucidated. In addition, lactate, inosine, and hypoxanthine, measured in the ECF by intracerebral microdialysis, may prove to have diagnostic and/or prognostic value in neurometabolic monitoring of the ischemic brain.

Calcium movements in traumatic brain injury: the role of glutamate receptor-operated ion channels.

Ion-selective microelectrodes were used to study acute effects of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy- 5-methyl-4-isoxazole (AMPA) receptor blockade on posttraumatic calcium disturbances. An autoradiographic technique with 45 Ca2+ was used to study calcium disturbances at 8, 24, and 72 h. Compression contusion trauma of the cerebral cortex was produced by a 21-g weight dropped from a height of 35 cm onto a piston that compressed the brain 2 mm. Pre- and posttrauma interstitial [Ca2+] ([Ca2+]e) concentrations were measured in the perimeter, i.e., the shear stress zone (SSZ) and in the central region (CR) of the trauma site. For the [Ca2+]e studies the animals were divided into controls and groups pretreated with dizocilipine maleate (MK-801) or with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[F]quinoxaline (NBQX). In all groups, [Ca2+]e decreased from pretrauma values of approximately 1 mM to posttraumatic values of 0.1 mM in both the CR and the SSZ. This was followed by a slow restitution toward pretraumatic levels during the 2-h observation period. There was no significant difference in recovery pattern between controls and pretreated animals. Accumulation of 45Ca2+ and serum proteins was seen in the entire SSZ, while neuronal necrosis was confined to a narrow band within the SSZ. The CR was unaffected apart from occasional eosinophilic neurons and showed no accumulation of 45Ca2+. Posttraumatic treatment with MK-801 or NBQX had no obvious effect on neuronal injury in the SSZ. We conclude that (a) acute [Ca2+]e disturbances in compression contusion brain trauma are not affected by blockade of NMDA or AMPA receptors, (b) 45Ca2+ accumulation in the SSZ reflects mainly protein accumulation due to blood-brain barrier breakdown rather than cell death, and (c) acute cellular Ca2+ over-load per se does not seem to be a major determinant of cell death after cerebral trauma in our model.

The effect of MK-801 on cortical spreading depression in the penumbral zone following focal ischaemia in the rat.

Cortical spreading depression (CSD) is a transient depression of neuronal activity that spreads across the cortical surface. In the present studies, we have investigated CSD activity in the penumbral zone following permanent middle cerebral artery (MCA) occlusion in the rat (n = 16/group), using double-barreled Ca(2+)-sensitive microelectrodes. Measurements of CSD activity were made for 3 h in each animal. During this time, a varying number of spontaneous CSDs were seen in the control group (total was 30, with a range of 0-7/rat). These CSDs were of varying duration: "small" (approximately 1 min) and "big" (5-45 min) CSDs. During a CSD, the extracellular [Ca2+] decreased to 0.11 +/- 0.07 mM (mean +/- SD). After 3 h, the extracellular [Ca2+] in the cortex (penumbral zone) was either normal (10/16 rats) or lowered to 0.5 mM (2/16 rats) or to 0.1 mM (4/16 rats). In the caudate nucleus (ischaemic core area), all rats had an extracellular [Ca2+] of approximately 0.1 mM when measured after the 3 h recording period. Neuropathological evaluation of the brains of the animals, which had been allowed to survive for 24 h after MCA occlusion, revealed ischaemic damage in the dorsolateral cortex and caudate nucleus. Administration of the noncompetitive NMDA antagonist, MK-801 (3 mg/kg i.p.), 30 min after MCA occlusion resulted in 24 and 29% reductions in the volume of hemispheric and cortical damage, respectively, which was highly significant (p less than 0.0001); no protection was seen against caudate damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial function and energy metabolism after hypoxia-ischemia in the immature rat brain: involvement of NMDA-receptors.

Treatment after hypoxia-ischemia (HI) in immature rats with the N-methyl-D-aspartate receptor (NMDAR) antagonist dizocilpine maleate (MK-801) reduces areas with high glucose utilization and reduces brain damage. The object was to study the metabolic effects of MK-801 treatment after HI. Seven-day-old rats were randomized to the following groups: non-HI, HI, or HI plus MK-801 (0.5 mg/kg immediately after HI). In the parietal cortex, the mitochondrial respiration was measured in homogenates 1 to 4 hours, and the energy metabolites at 3 and 8 hours after HI. The energy use was calculated from changes in energy metabolites after decapitation at 3 hours after HI. State 3 respiration was reduced by 46%, 32%, and 25% after HI compared with non-HI with pyruvate plus malate, glutamate plus malate, or glutamate plus succinate as substrates, respectively. Uncoupler-stimulated but not state 4 respiration was similarly reduced. The MK-801 augmented pyruvate plus malate-supported state 3 respiration after HI by 42%. The energy utilization was not affected by HI but was reduced by MK-801 treatment in the ipsilateral cortex from 4.6 +/- 2.3 to 2.6 +/- 1.8 micromol high-energy phosphate bond/min/g. The levels of ATP and phosphocreatine did not differ between the HI and HI plus MK-801 groups at 3 hours, but were lower in the HI than in the HI plus MK-801 group at 8 hours after HI. In conclusion, treatment with MK-801 reduced energy utilization and improved mitochondrial function and energy status after HI, suggesting a linkage between NMDAR activation and impaired energy metabolism during reperfusion.

Intracerebral microdialysis of extracellular amino acids in the human epileptic focus.

Extracellular levels of aspartate (ASP), glutamate (GLU), serine (SER), asparagine (ASN), glycine (GLY), threonine (THR), arginine (ARG), alanine (ALA), taurine (TAU), tyrosine (TYR), phenylalanine (PHE), isoleucine (ILEU), and leucine (LEU) were monitored by using intracerebral microdialysis in seven patients with medically intractable epilepsy, undergoing epilepsy surgery. In association with focal seizures, dramatic increases of the extracellular ASP, GLU, GLY, and SER concentrations were observed. The other amino acids analyzed, including TAU, showed small changes. The results support the hypothesis that ASP, GLU, GLY, and possibly SER, play an important role in the mechanism of seizure activity and seizure-related brain damage in the human epileptic focus.

Paradoxical increase in neuronal DNA fragmentation after neuroprotective free radical scavenger treatment in experimental traumatic brain injury.

The mechanisms and role of nerve cell death after traumatic brain injury (TBI) are not fully understood. The authors investigated the effect of pretreatment with the oxygen free radical spin trap alpha-phenyl-N-tert-butyl-nitrone (PBN) on the number of neurons undergoing apoptosis after TBI in rats. Apoptotic cells were identified by the TUNEL method combined with the nuclear stain, Hoechst 33258, and immunohistochemistry for the active form of caspase-3. Numerous neurons became positive for activated caspase 3 and TUNEL in the cortex at 24 hours after injury, suggesting ongoing biochemical apoptosis. In PBN-treated rats, a significantly greater number of cells were found to be TUNEL positive at 24 hours compared with controls. However, PBN treatment resulted in a reduced cortical lesion volume and improved behavioral outcome two weeks after injury. The authors conclude that a treatment producing an increase in DNA fragmentation in the early phase may be compatible with an overall beneficial effect on outcome after TBI. This should be considered in the screening process for future neuroprotective remedies.

Regional changes in interstitial K+ and Ca2+ levels following cortical compression contusion trauma in rats.

Brain trauma is associated with acute functional impairment and neuronal injury. At present, it is unclear to what extent disturbances in ion homeostasis are involved in these changes. We used ion-selective microelectrodes to register interstitial potassium ([K+]e) and calcium ([Ca2+]e) concentrations in the brain cortex following cerebral compression contusion in the rat. The trauma was produced by dropping a 21 g weight from a height of 35 cm onto a piston that compressed the cortex 1.5 mm. Ion measurements were made in two different locations of the contused region: in the perimeter, i.e., the shear stress zone (region A), and in the center (region B). The trauma resulted in an immediate increase in [K+]e from a control level of 3 mM to a level > 60 mM in both regions, and a concomitant negative shift in DC potential. In both regions, there was a simultaneous, dramatic decrease in [Ca2+]e from a baseline of 1.1 mM to 0.3-0.1 mM. Interstitial [K+] and the DC potential normalized within 3 min after trauma. In region B, [Ca2+]e recovered to near control levels within 5 min after ictus. In region A, however, recovery of [Ca2+]e was significantly slower, with a return to near baseline values within 50 min after trauma. The prolonged lowering of [Ca2+]e in region A was associated with an inability to propagate cortical spreading depression, suggesting a profound functional disturbance. Histologic evaluation 72 h after trauma revealed that neuronal injury was confined exclusively to region A. The results indicate that compression contusion trauma produces a transient membrane depolarization associated with a pronounced cellular release of K+ and a massive Ca2+ entry into the intracellular compartment. We suggest that the acute functional impairment and the subsequent neuronal injury in region A is caused by the prolonged disturbance of cellular calcium homeostasis mediated by leaky membranes exposed to shear stress.

Simultaneous intracerebral microdialysis and positron emission tomography in the detection of ischemia in patients with subarachnoid hemorrhage.

Intracerebral microdialysis (MD) was applied in patients with subarachnoid hemorrhage. The regional CBF, the CMRO2, and oxygen extraction ratio (OER) were measured with simultaneous positron emission tomography (PET). The aim was to directly correlate alterations in dialysate levels of energy-related metabolites (lactate, lactate/pyruvate ratio, hypoxanthine) and excitatory amino acids (EAAs) (glutamate and aspartate) to the energy state in the MD probe region as determined by PET. Regional ischemia was defined according to Heiss et al. and Lassen (Heiss et al., 1992; Lassen, 1966). Whole-brain ischemia was considered present when the OER for the whole brain exceeded the mean whole-brain OER + 2 SD of six reference patients. In general, the presence of whole-brain ischemia and/or regional ischemia within the region of the MD probe was associated with increased levels of energy-related metabolites and EAAs retrieved by MD. Increased levels of energy-related metabolites and EAAs were only occasionally seen when PET did not show any signs of ischemia or when signs of regional ischemia were found remote from the MD probe region. Thus, the energy-related metabolites and EAAs may be used as extracellular "markers" of ischemia. PET may be of use in defining critical ischemic regions (tissue at risk) where the MD probe can be inserted for chemical monitoring.

Mn2+ prevents the Ca2+-induced inhibition of ATP synthesis in brain mitochondria.

Uptake of Ca2+ by rat brain mitochondria causes an inhibition of respiratory stimulation by ADP, and the inhibition is relieved upon Na+-induced release of Ca2+ from the mitochondria, in accordance with earlier reports. We show that simultaneous uptake of Ca2+ and Mn2+ results in no inhibition of ADP-stimulated respiration, indicating that Mn2+ prevents the Ca2+-induced inhibition of ATP synthesis, without preventing Ca2+ accumulation in the mitochondria. The results are discussed in relation to a possible involvement of the mitochondrial ATPase-inhibitor protein in the observed effects of Ca2+ and Mn2+.

Regulation of pituitary adenylate cyclase activating polypeptide and its receptor type 1 after traumatic brain injury: comparison with brain-derived neurotrophic factor and the induction of neuronal cell death.

Neurotrophic factors are known to promote neuronal survival during development and after acute brain injury. Recent data suggest that some neuropeptides also exhibit neurotrophic activities, as shown for the pituitary adenylate cyclase activating polypeptide, which increases the survival of various neuronal populations in culture. Employing in situ hybridization techniques, we have studied the regulation of messenger RNA for pituitary adenylate cyclase activating polypeptide and its receptor type 1 after a moderate traumatic brain injury to rat brain cortex. We have further compared their messenger RNA expression to that of brain-derived neurotrophic factor and to the amount of cell death occurring in the brain at various times after the brain injury. Levels of brain-derived neurotrophic factor messenger RNA increased rapidly within 2 h after trauma in cortex and hippocampus, and returned to control levels thereafter. The levels of messenger RNA for pituitary adenylate cyclase activating polypeptide also increased with time in the injured brains and reached maximal expression at 72 h, i.e. the end of the observation period. The alterations in pituitary adenylate cyclase activating polypeptide messenger RNA levels were particularly pronounced in the perifocal region and in the ipsilateral dentate gyrus of the brain injury. In contrast, the messenger RNA levels encoding pituitary adenylate cyclase activating polypeptide receptor type 1 first decreased after trauma and were then normalized in the dentate gyrus. There was a large increase in the number of cells labelled for DNA breaks at 12 h post-trauma, indicative of enhanced cell death. The number of labelled cells, however, decreased at later stages concomitant with an increase in the expression of pituitary adenylate cyclase activating polypeptide messenger RNA. Pituitary adenylate cyclase activating polypeptide rescued cortical neurons in cultures against ionomycin-induced cell death, supporting the concept of a neuroprotective effect for the peptide. These results demonstrate a differential regulation of messenger RNA for brain-derived neurotrophic factor and the pituitary adenylate cyclase activating polypeptide and its receptor after brain trauma. The data also suggest that pituitary adenylate cyclase activating polypeptide might have a beneficial effect in brain injury by counteracting neuronal cell death.