Double versus single intensive phototherapy with LEDs in treatment of neonatal hyperbilirubinemia.

OBJECTIVE: We investigate whether double phototherapy reduces total serum bilirubin concentration faster than single light during intensive phototherapy with high levels of irradiance using light-emitting diodes. STUDY DESIGN: Eighty-three infants with gestational age ⩾33 weeks and uncomplicated hyperbilirubinemia were randomized to either double (n=41) or single phototherapy (n=42) for 24 h. The mean irradiance was 64.8 µW cm-2 nm-1 from above and 39 µW cm-2 nm-1 from below. RESULTS: The percentage decreases of total serum bilirubin after 12 h of double vs single phototherapy were (mean (95% confidence interval (CI))) 39% (37 to 42) vs 30% (27 to 32), respectively (P<0.001). After 24 h, the decreases were 58% (56 to 61) vs 47% (44 to 50), respectively (P<0.001). The results were still significant after adjustment for confounding. The only side effect was loose stools. CONCLUSION: Even with intensive phototherapy increasing spectral power by increasing the irradiated body surface area, the efficacy of phototherapy is improved.

Extremely prematurely born adolescents self-report of anxiety symptoms, and the mothers' reports on their offspring.

AIM: To compare anxiety symptoms in adolescents born extremely prematurely to term-born controls. METHODS: We had 96 preterm-born adolescents and 40 term-born controls from Denmark, and their mothers score the adolescents on the Revised Children Anxiety and Depression scale. We analysed group differences, cross-informant correlations and relative risks for elevated anxiety symptoms. RESULTS: Self-reported anxiety symptoms did not significantly differ, although the upper confidence limit (95% CI: -3.3 to 5.1) supported an odds ratio of 2 for the preterm-born participants. Mothers of the preterm-born participants reported higher social anxiety symptoms than did mothers of controls (51.7 versus 46.8, p = 0.001). The relative risk for being above a threshold indicating distressing anxiety was small from self-reports (1.39; p = 0.60). From mother-reports, the relative risk was noticeable but not significant (4.58; p = 0.14). Cross-informant scores correlated significant for total anxiety and social anxiety for the preterm-born (rτ = 0.2, p = 0.001; rτ = 0.3, p ≤ 0.001). CONCLUSIONS: Self-reports did not clearly indicate more anxiety in the preterm group, although confidence intervals supported a possible twofold increase. Mother- and self-reports correlated only for the preterm group, which may indicate increased sensitivity for their children's symptoms.

An age difference of two billion years between a metal-rich and a metal-poor globular cluster.

Globular clusters trace the formation history of the spheroidal components of our Galaxy and other galaxies, which represent the bulk of star formation over the history of the Universe. The clusters exhibit a range of metallicities (abundances of elements heavier than helium), with metal-poor clusters dominating the stellar halo of the Galaxy, and higher-metallicity clusters found within the inner Galaxy, associated with the stellar bulge, or the thick disk. Age differences between these clusters can indicate the sequence in which the components of the Galaxy formed, and in particular which clusters were formed outside the Galaxy and were later engulfed along with their original host galaxies, and which were formed within it. Here we report an absolute age of 9.9 ± 0.7 billion years (at 95 per cent confidence) for the metal-rich globular cluster 47 Tucanae, determined by modelling the properties of the cluster's white-dwarf cooling sequence. This is about two billion years younger than has been inferred for the metal-poor cluster NGC 6397 from the same models, and provides quantitative evidence that metal-rich clusters like 47 Tucanae formed later than metal-poor halo clusters like NGC 6397.

Increased nuchal translucency, normal karyotype and infant development.

OBJECTIVE: To investigate whether chromosomally normal fetuses with a nuchal translucency (NT) ≥ 99th percentile(3.5 mm) in the first trimester have an increased risk of delayed development at 2 years of age. METHODS: The study included children of women from 10 Danish hospitals who had fetal NT either ≥ 99th percentile (Group 1) or <95th percentile (Group 2) in the first trimester. The groups were matched by gender,gestational age at birth and maternal age. There were twice as many children in Group 2 as in Group 1. Follow-up was conducted at 2 years of age, infant development being assessed by the 'Ages and Stages Questionnaire'. The cutoff value for delayed development was defined as the 5th percentile from the first 100 questionnaires from Group 2. RESULTS: In a 1-year period 202 of 33 266 fetuses (0.6%)had NT ≥ 3.5 mm. Of these, 99 (49.0%) were liveborn with normal karyotype and normal ultrasound findings during pregnancy. The response rate to the ASQ was 83.3% in Group 1 and 71.4% in Group 2. A low ASQ score was found in 1.3% (1/80) and 4.4% (6/137)in Groups 1 and 2, respectively (P = 0.265), but no difference was found in the mean ASQ score between the two groups (P = 0.160). CONCLUSION: Fetuses with NT ≥ 99th percentile, normal karyotype and normal ultrasound findings during pregnancy had no increased risk of developmental delay at 2 years of age compared with fetuses with normal NT(<95th percentile).

The members of the Bacillus cereus group are commonly present contaminants of fresh and heat-treated milk.

To determine the level of milk contamination by Bacillus cereus sensu lato, 44 samples were collected from a dairy farm and two independent dairies in northeastern Poland. A total of 680 B. cereus sensu lato isolates were recovered. Based on spore counts, their highest level in milk was found during the spring and summer months. Although significant variations in chromosomal DNA polymorphisms among B. cereus sensu lato isolates were noted based on repetitive element sequence polymorphism (rep-PCR) and pulse-field gel electrophoresis (PFGE), indistinguishable B. cereus isolates were observed in all sampling points and locations. Both B. cereus sensu stricto/Bacillus weihenstephanensis and Bacillus thuringiensis cultured from milk harbored nheA, hblA, and cytK in, respectively, 80%, 55%, and 60% of the isolates. With respect to Bacillus mycoides/Bacillus pseudomycoides, 30% and 70% of theses isolates harbored, respectively, nheA and hblA. The presence of cytK was not detected in any isolate. Our data show the occurrence of potentially toxic B. cereus s.l. in both raw and heat-treated milk, thus emphasizing the requirement for precautions that prevent spore germination and vegetative proliferation by keeping the milk at low temperatures during all steps of production and dispensation to the consumers.

Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats.

AIMS: To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats. METHODS AND RESULTS: Germination was studied by feeding germ-free rats with spores of a B. thuringiensis strain harbouring a plasmid encoding green fluorescent protein (GFP), which enabled quantification of germinated bacteria by flow cytometry. To study in vivo conjugation, germ-free rats were first associated with a B. thuringiensis recipient strain and after 1 week an isogenic donor strain harbouring the conjugative plasmid pXO16 was introduced. Both strains were given as spores and transfer of pXO16 was observed from the donor to the recipient strain. CONCLUSIONS: Bacillus thuringiensis is able to have a full life cycle in the intestine of gnotobiotic rats including germination of spores, several cycles of growth and sporulation of vegetative cells. For the first time conjugative plasmid transfer in a mammalian intestinal tract was shown between two B. thuringiensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of B. thuringiensis are used worldwide to combat insect pests, and this study brings new insights into the nature of B. thuringiensis showing the potential of the bacteria to germinate and transfer DNA in the mammalian intestinal tract.

Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9.

AIMS: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers. METHODS AND RESULTS: Bacillus thuringiensis ssp. entomocidus HD9 produced in the culture supernatant an antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes, one of four pathogenic Pseudomonas aeruginosa and several fungi. Production of the antibacterial activity, named entomocin 9, started during mid-logarithmic growth reaching its maximum at the early stationary phase. Entomocin 9 retained more than 72% of activity after incubation for 20 min at 121 degrees C. Activity was lost after proteinase K treatment, it was stable in a pH range between 3 and 9, and resistant to lyophilization. After partial purification with ammonium sulphate precipitation followed by gel-filtration and anion-exchange chromatography, an active protein of ca 12.4 kDa was isolated. The mode of action of entomocin 9 was bactericidal and caused cell lysis of growing cells. Despite the presence of a range of virulence related genes, including haemolysin BL, nonhaemolytic enterotoxin, cytotoxin K and several hydrolytic activities, B. thuringiensis HD9 was not toxic against Vero cells. CONCLUSIONS: Entomocin 9 is a novel heat-stable, bacteriocin produced by B. thuringiensis HD9. The absence of toxicity against Vero cells suggests the suitability of strain HD9 for a safe application in antimicrobial treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: New finding on entomocin 9 would make B. thuringiensis attractive in biotechnological applications as an antimicrobial agent in agriculture and food industry.

The hidden lifestyles of Bacillus cereus and relatives.

Bacillus cereus sensu lato, the species group comprising Bacillus anthracis, Bacillus thuringiensis and B. cereus (sensu stricto), has previously been scrutinized regarding interspecies genetic correlation and pathogenic characteristics. So far, little attention has been paid to analysing the biological and ecological properties of the three species in their natural environments. In this review, we describe the B. cereus sensu lato living in a world on its own; all B. cereus sensu lato can grow saprophytically under nutrient-rich conditions, which are only occasionally found in the environment, except where nutrients are actively collected. As such, members of the B. cereus group have recently been discovered as common inhabitants of the invertebrate gut. We speculate that all members disclose symbiotic relationships with appropriate invertebrate hosts and only occasionally enter a pathogenic life cycle in which the individual species infects suitable hosts and multiplies almost unrestrained.

Polymerase chain reaction assay for the detection of Bacillus cereus group cells.

Recent investigations have shown that members of the Bacillus cereus group carry genes which have the potential to cause gastrointestinal and somatic diseases. Although most cases of diseases caused by the B. cereus group bacteria are relatively mild, it is desirable to be able to detect members of the B. cereus group in food and in the environment. Using 16S rDNA as target, a PCR assay for the detection of B. cereus group cells has been developed. Primers specific for the 16S rDNA of the B. cereus group bacteria were selected and used in combination with consensus primers for 16S rDNA as internal PCR procedure control. The PCR procedure was optimized with respect to annealing temperature. When DNA from the B. cereus group bacteria was present, the PCR assay yielded a B. cereus specific fragment, while when non-B. cereus prokaryotic DNA was present, the consensus 16S rDNA primers directed synthesis of the PCR products. The PCR analyses with DNA from a number of non-B. cereus confirmed the specificity of the PCR assay.

Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis.

Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereus and B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceT gene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes in B. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.

Natural coprevalence of Strongwellsea castrans, Cystosporogenes deliaradicae, and Bacillus thuringiensis in the host, Delia radicum.

Adult cabbage root flies (Delia radicum) from three Danish localities were diagnosed microscopically for the natural prevalence of Strongwellsea castrans, Cystosporogenes deliaradicae, and Bacillus thuringiensis. C. deliaradicae was significantly coprevalent with S. castrans. B. thuringiensis sporangia were diagnosed in the hemolymph in two D. radicum which were also infected with S. castrans and proved to belong to serovar aizawai and serovar balearica. The biological characterization of S. castrans proved that at 17.5 degrees C flies developed an abdominal hole 7.9 days (mean) after infection and that 5.7 days (mean) passed from the emergence of the hole to the death of the infected host. No mortality effect among D. radicum subjected to B. thuringiensis serovar aizawai, balearica, and kurstaki isolates was detected. RAPD with DNA proved that six B. thuringiensis serovar balearica isolates (all from the same fly) were indistinguisable. This indicates that proliferation of B. thuringiensis in the abdomen of an S. castrans-infected D. radicum may be due to just one genotype. The profiles of one isolated aizawai strain did not correspond to the profiles of other serovar aizawai strains used for comparison. The biological significance of the interaction between the involved pathogens is discussed.

Genome stability of Bacillus thuringiensis subsp. israelensis isolates.

Swedish soil isolates biochemically classified as Bacillus thuringiensis subsp. israelensis were further examined for genetic diversity by multilocus enzyme electrophoresis (MLEE), random amplified polymorphic DNA analysis (RAPD), pulse field gel electrophoresis (PFGE), and Southern blotting, and were compared with reference strains. All the tested strains belonging to the Bt. israelensis serotype H14 were found to be identical, as judged from the RAPD analysis. MLEE analysis gave a similar result; only one H14 strain was found to differ from the remaining H14 strains by one null allele. PFGE analysis confirmed a very close relationship between the H14 strains but revealed an SfiI restriction fragment of variable size. Southern blot analyses were carried out with probes for the chromosomally encoded flagellin gene(s) and the plasmid-encoded mosquitocidal toxins. All probes gave similar hybridization patterns in the H14 strains. The mosquito toxin probes hybridized only to the H14 strains, except for one probe hybridizing to strain 6:3, which was originally isolated from the same soil sample as strains 6:11 and 6:12. Because the RAPD, MLEE, and PFGE analyses showed that strain 6:3 appears to be unrelated to strains 6:11 and 6:12, the presence of a mosquito toxin sequence in strain 6:3 may suggest that gene transfer has occurred.

Hypoxia-tolerant neonatal CA1 neurons: relationship of survival to evoked glutamate release and glutamate receptor-mediated calcium changes in hippocampal slices.

Neurons in the neonatal mammalian brain survive greater degrees of hypoxic stress than those in the mature brain. To investigate how developmental changes in glutamate receptor-mediated neurotoxicity contribute to this difference, we measured hypoxia-evoked glutamate release, glutamate receptor contribution to hypoxia-evoked intracellular calcium changes, and survival of hypoxia-/ischemia-sensitive CA1 neurons in rat hippocampus. Glutamate release was measured by a fluorescence assay, calcium changes in CA1 neurons with fura-2, and cell viability using Nissl and fluorescence staining with calcein-AM/ethidium homodimer, all in 300-micron thick hippocampal slices from 3-30 post-natal day (PND) rats. Glutamate released from PND 3-7 slices during hypoxia (PO2 = 5 mmHg) was only one third that of PND 18-22 slices. In PND 3-7 slices, survival of CA1 neurons after 5 min of hypoxia and 6 h of recovery was significantly greater than in PND 18-22 slices (viability indices 0.60 and 0.28, respectively, (p < 0.05). Five min of anoxia significantly altered Nissl staining pattern and morphology of CA1 neurons in PND 18-22 but not PND 3-7 slices. Hypoxia (PO2 = 5 mm Hg) caused three to five times greater increases in [Ca2+]i in PND 18-22 slices than in PND 3-7 slices (p < 0.001). During re-oxygenation, [Ca2+]i returned to baseline in PND 3-7 slices, but remained elevated in PND 18-22 slices. Glutamate receptor-mediated calcium changes in CA1 during hypoxia were 33% and 62% of the total calcium change in PND 3-7 and PND 18-22 CA1, respectively. We conclude that survival of CA1 neurons in PND 3-7 slices following hypoxic stress is associated with smaller increases and enhanced recovery of [Ca2+]i, less accumulation of glutamate, and less glutamate receptor-mediated calcium influx than in PND 18-22 slices.

Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.

Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as delta9-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (deltaCa2+NMDA) in rat brain slices. The presence of anandamide reduced deltaCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting deltaCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences deltaCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated deltaCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.

Natural occurrence of Bacillus thuringiensis on cabbage foliage and in insects associated with cabbage crops.

Bacillus thuringiensis was isolated from the phylloplane of organically grown cabbage in one field during two growth seasons (1992-93). The frequency of B. thuringiensis varied between 0.02 and 0.67 of the total B. cereus/B. thuringiensis population, with an average of 0.11. Characterization of the B. thuringiensis isolates from foliage showed that the majority (64% of 150 isolates) belonged to serovar kurstaki, had bipyramidal crystals and toxicity towards Pieris brassicae and/or Trichoplusia ni. Other serovars were also found on the foliage but occurred at very low frequencies (one to three isolates of each serovar). Bacillus thuringiensis was also isolated from insects associated with the cabbage crop (Pieris rapae (Lep.), Delia radicum (Dip.), Syrphidae ribesii (Dip.) and Aleochara bilineata (Col.)), which were collected alive at different developmental stages in the same field. Serologically these isolates were assigned to the serovars kurstaki, aizawai, tochigiensis, colmeri and indiana/colmeri.

Enterotoxin-producing strains of Bacillus thuringiensis isolated from food.

Strains of Bacillus thuringiensis were isolated from various food items (pasta, pitta bread and milk) and were found to belong to either H-serotype kurstaki or neoleonensis. The strains were bioassayed against Pieris brassicae and insecticidal activity of strains was found to correspond to the presence of the cry1A-gene. All strains, except one, were found to express cytotoxic effects on Vero cells as an indicator of enterotoxin activity. Further, the B. thuringiensis strains HD-1 (serotype kurstaki), NB-125 (serotype tenebrionis) and HD-567 (serotype israelensis ) which are used commercially for insect pest management, were also found to have cytotoxic effects on Vero cells.

Alpha 2-adrenergic agonists reduce glutamate release and glutamate receptor-mediated calcium changes in hippocampal slices during hypoxia.

The mechanisms by which alpha 2-adrenergic agonists reduce ischemic brain damage are not clear. In ischemia-vulnerable hippocampal neurons we tested whether alpha 2-agonists reduce glutamate efflux and glutamate receptor-mediated increase of cytosolic free calcium. Brain slices (300 microns thick) from rat hippocampal were located with fura-2 for measurements of cytosolic free calcium with a microscope fluorometer. Change of cytosolic calcium in CA1 neurons during application of N-methyl-D-aspartate (NMDA) was measured, as were calcium changes during simulated ischemia (hypoxia, NaCN, iodoacetate) of hypoxia plus high glutamate concentration (pO2 = 25 mmHg, 3 mM glutamate). In order slices, glutamate efflux evoked by anoxia (pO2 = 25 mmHg, 100 microM NaCN) was measured. The selective alpha 2-agonist mivazerol (1 microM) decreased NMDA receptor-mediated calcium changes in hippocampal CA1 neurons by 28% (p = 0.0079). With hypoxia and 3 mM glutamate, 1 microM mivazerol reduced early peak calcium changes in CA1 neurons by 57% (p = 0.0007). An alpha 2-antagonist (rauwolscine, 1 microM) blocked this. Mivazerol did not reduce the rate of calcium change during simulated ischemia. Clonidine (0.1 microM), a partial alpha 2-agonist, decrease glutamate/hypoxia-mediated calcium changes in CA1 (p = 0.01), but 1 microM clonidine, which stimulates alpha 1-receptors, did not. Mivazerol decreased hypoxia and KCl1-evoked glutamate release by 50% and 75% (p < 0.01), respectively. In addition, 1 microM mivazerol reduced lactate dehydrogenase leakage rate from brain slices during anoxia by 61% (p = 0.018). Thus, alpha 2-receptors influence glutamate release, calcium changes, and cell damage in ischemia-vulnerable hippocampal neurons. These effects may contribute to the cerebroprotective actions of alpha 2-agonists.

Influence of compensated radioiodine therapy on thyroid volume and incidence of hypothyroidism in Graves' disease.

OBJECTIVES: To investigate the long-term effect of radioactive iodine (131I) on thyroid function and size in patients with Graves' disease. SETTING: Out-patient clinic in Herlev Hospital. SUBJECTS: One hundred and seventeen consecutive patients (104 women) with Graves' disease selected for 131I treatment and followed for a minimum of 12 months (range 1-10 years, median 5 years). INTERVENTIONS: 131I dose was calculated based on thyroid volume and 24-h 131I uptake. MAIN OUTCOME MEASURES: Standard thyroid function variables and ultrasonically determined thyroid volume before treatment as well as 0.75, 1.5, 3, 6 and 12 months after treatment, and then once a year were investigated. RESULTS: Seventy-eight patients were cured by one 131I dose and 30 by two doses, while the remaining nine patients received additional doses (range one to five doses, median one dose). Within one year, 25% developed hypothyroidism, and hereafter, hypothyroidism developed at a constant rate of 3% per year independent of antithyroid pretreatment. The cumulative 10-year risk of hypothyroidism was 60%. Initial median thyroid volume was 33 mL (range 9-106 mL). At 12 months after the last 131I dose, median thyroid volume was reduced to 14 mL (range 6-36 mL) (P < 0.00001). The median reduction being 58% (range 0-80%), hereafter no further reduction occurred. A significant reduction in thyroid volume was also noted in patients needing subsequent 131I doses and in those developing hypothyroidism within the first year. CONCLUSIONS: 131I normalizes thyroid volume in patients with Graves' disease. Hypothyroidism seems an inevitable end result of this treatment. The present study suggests that it will be impossible to modify 131I therapy in a way to achieve both early control of hyperthyroidism and a low incidence of hypothyroidism.

Effects of isoflurane and hypothermia on glutamate receptor-mediated calcium influx in brain slices.

BACKGROUND: To understand how volatile anesthetics protect neurons during cerebral ischemia, we studied the effects of isoflurane on cerebral glutamate receptor-mediated calcium influx. Calcium influx via these key excitatory receptors may mediate pain transmission, memory, and the pathophysiologic sequelae of cerebral anoxia or ischemia. Because cerebral protection by hypothermia may involve a decrease in glutamate receptor activity, we also examined the interaction of temperature and isoflurane on glutamate receptor inhibition. METHODS: We measured glutamate receptor-mediated changes in cytosolic calcium in 300-microns-thick rat cortical brain slices. Temperature was varied to 28, 34, 37, or 39 degrees C and isoflurane partial pressure to 0.016-0.019 atm (equivalent to 1.16 minimum alveolar concentration [MAC], adjusted for temperature and age). Brain slices were loaded with fura-2 to permit measurement of cytosolic free calcium. Calcium changes due to the glutamate receptor agonist N-methyl-D-aspartate (NMDA) (50 microM), to ischemia levels of L-glutamate (1.0 mM) or to simulated ischemia (1.0 mM glutamate, 100 microM NaCN, and 3.5 mM iodoacetate) was then measured. Slice lactate dehydrogenase leakage and adenosine triphosphate were measured as indices of cellular integrity. RESULTS: Isoflurane reduced both L-glutamate and NMDA-mediated calcium fluxes by approximately 60%. Neither the activity of the NMDA receptor nor its inhibition by isoflurane was altered by temperature. The rate of calcium influx during ischemia was significantly reduced both by temperature and by isoflurane (P < 0.05). Adenosine triphosphate loss and lactate dehydrogenase leakage were reduced by isoflurane during simulated ischemia by 37% and 73% (P < 0.05), respectively. CONCLUSIONS: (1) At 1.16 MAC, isoflurane potently inhibits glutamate receptors and delays cellular injury induced by simulated ischemia, and (2) hypothermia does not reduce the intrinsic activity of cortical glutamate receptors but delays calcium accumulation during simulated ischemia. Isoflurane reduces the severity of key pathophysiologic events in an in vitro model of simulated cerebral ischemia.

Causes of calcium accumulation in rat cortical brain slices during hypoxia and ischemia: role of ion channels and membrane damage.

To better understand why neurons accumulate calcium during cerebral ischemia, the influence of specific ion channel inhibitors on the rise in cytosolic free calcium ([Ca2+]c) during hypoxia or ischemia was evaluated in rat cerebrocortical brain slices. [Ca2+]c was measured fluorometrically with the dye fura-2 during hypoxia (95% N2/5% CO2 or 100 microM NaCN), simulated ischemia (100 microM NaCN plus 3.5 mM iodoacetate), or 0.5-1.0 mM glutamate. Hypoxia or ischemia increased [Ca+2]c from 100-250 nM to 1,000-2,500 nM within 3-5 min. Greater than 85% of the calcium accumulation was influx from the extracellular medium. The non-competitive N-methyl-D-aspartate (NMDA) inhibitor MK-801 reduced [Ca2+]c accumulation during hypoxia, but antagonism of alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptors or voltage-gated sodium or calcium channels or Na+/Ca2+ exchangers had no effect. During ischemia, combined antagonism of NMDA, AMPA and voltage-gated sodium channels slowed the rate of calcium accumulation, but not concentration at 5 min. Membrane damage, as indicated by leakage of lactate dehydrogenase into superfusate, occurred coincidentally with calcium influx and ATP loss during both hypoxia and ischemia. We conclude that cytosolic calcium changes during hypoxia or ischemia in cortical brain slices are due to multiple mechanisms, are incompletely inhibited by combined ion channel blockade, and are associated with disruption of cell membrane integrity.