Notes: Abstract: Compounds known to affect glycogen metabolism in vivo or in cell-free preparations were used to investigate the regulation of glycogen content in intact astrocytes cultured from newborn rat cortex. Compounds were added with fresh medium to culture dishes, and astrocyte glucose and glycogen content determined 24 h later. Increasing the medium glucose concentration from 7.5 mM to 30 mM increased cell glycogen content 80%. Addition of 2-deoxyglucose or 3-O-methyl glucose (2.5–10 mM) also increased cell glycogen content, 50–100%, suggesting a regulatory rather than mass action effect of glucose on astrocyte glycogen content. The phosphorylase b inhibitors 2,2′,4,4′,5,5′-hexabromobiphenyl and riboflavin had no effect on astrocyte glycogen content, consistent with negligible phosphorylase b activity in normal astrocytes. Phenobarbital and l-methionine-DL-sulfoximine (MSO) are both known to induce astrocyte glycogen accumulation in vivo. The addition of phenobarbital (2 mM) had no effect on the glycogen content of cultured astrocytes, suggesting an indirect mechanism for the in vivo effect. MSO at 1 mM, however, induced a 300% increase in glycogen content. The time course of glucose and glycogen content after MSO administration suggests this increase to be the result of slowed glycogenolysis rather than accelerated glycogen synthesis.

Notes: Abstract The effects of arachidonic acid on glutamate and γ-aminobutyric acid (GABA) uptake were studied in primary cultures of astrocytes and neurons prepared from rat cerebral cortex. The uptake rates of glutamate and GABA in astrocytic cultures were 10.4 nmol/mg protein/min and 0.125 nmol/mg protein/min, respectively. The uptake rates of glutamate and GABA in neuronal cultures were 3.37 nmol/mg protein/min and 1.53 nmol/mg protein/min. Arachidonic acid inhibited glutamate uptake in both astrocytes and neurons. The inhibitory effect was observed within 10 min of incubation with arachidonic acid and reached approximately 80% within 120 min in both types of culture. The arachidonic acid effect was not only time-dependent, but also dose-related. Arachidonic acid, at concentrations of 0.015 and 0.03 μmol/mg protein, significantly inhibited glutamate uptake in neurons, whereas 20 times higher concentrations were required for astrocytes. The effects of arachidonic acid were not as deleterious on GABA uptake as on glutamate uptake in both astrocytes and neurons. In astrocytes, GABA uptake was not affected by any of the doses of arachidonic acid studied (0.015–0.6 μmol/mg protein). In neuronal cultures, GABA uptake was inhibited, but not to the same degree observed with glutamate uptake. Lower doses of arachidonic acid (0.03 and 0.015 μmol/mg protein) did not affect neuronal GABA uptake. Other polyunsaturated fatty acids, such as docosahexaenoic acid, affected amino acid uptake in a manner similar to arachidonic acid in both astrocytes and neurons. However, saturated fatty acids, such as palmitic acid, exerted no such effect. The significance of the arachidonic acid-induced inhibition of neurotransmitter uptake in cultured brain cells in various pathological states is discussed.

Notes: The glycogen content of primary cultured astrocytes was approximately doubled by incubation with 1 mM L-glutamate or L-aspartate. Other amino acids and excitatory neurotransmitters were without effect. The increase in glycogen level was not blocked by the glutamate receptor antagonist kynurenic acid but was completely blocked by the glutamate uptake inhibitor threo-3-hydroxy-D,L-aspartate and by removal of Na+ from the medium. Incubation with radiolabeled glucose and glutamate revealed that the increased glycogen content was derived almost entirely from glucose. Glutamate at 1 mM was also found to cause a 53 ± 12% decrease in glucose utilization and a 112 ± 69% increase in glucose-6-phosphate levels. These results suggest that the glycogen content of astrocytes is linked to the rate of glucose utilization and that glucose utilization can, in turn, be affected by the availability of alternative metabolic substrates. These relationships suggest a mechanism by which brain glycogen accumulation occurs during decreased neuronal activity.

Notes: Abstract: The effects of arachidonic acid and other polyunsaturated fatty acids (PUFAs) on both oxidative and metabolic perturbation were studied in primary cultures of rat cerebral cortical astrocytes. In the presence of 0.1 mM arachidonic acid, the rate of the reduction of nitroblue tetrazoiium (NBT) to nitroblue formazan (NBF) was stimulated from 0.65 ± 0.10 to 1.43 ± 0.15 and from 0.092 ± 0.006 to 0.162 ± 0.009 nmol/min/mg protein in intact and broken cell preparations, respectively. The rate of superoxide radical formation, as measured by the superoxide dismutase (SOD)-inhibitable NBT reduction was 0.042 nmol/mg protein in broken cells and was negligible in intact cells. The latter is due to the impermeability of SOD into the intact cell preparation. NBF formation in intact astrocytes stimulated by arachidonic acid was both time- and dosedependent. Other PUFAs, including linoleic acid, linolenic acid, and docosahexaenoic acid, were also effective in stimulating NBF formation in astrocytes, whereas saturated palmitic acid and monounsaturated oleic acid were ineffective. Similar effects of these PUFAs were observed in ma-londialdehyde formation in cells and lactic acid accumulation in incubation medium. These data indicate that both membrane integrity and cellular metabolism were perturbed by arachidonic acid and by other PUFAs. The sites of superoxide radical formation appeared to be intracellular and may be associated with membrane phospholipid domains, because liposome-entrapped SOD, which was taken up by intact astrocytes, reduced the level of superoxide radicals and lactic acid content, whereas free SOD was not effective.

Notes: Abstract: The mitochondrial toxin 3-nitropropionic acid (3-NP) produces selective striatal lesions in both experimental animals and humans. The pathogenesis of the lesions involves secondary excitotoxicity that may then lead to free radical generation. To test this further we examined the effects of 3-NP in both transgenic (Tg) mice that carry the complete sequence for the human copper/zinc superoxide dismutase (SOD) gene as well as non-Tg littermate controls. The Tg-SOD mice showed a pronounced attenuation of Nissl-stained striatal lesions compared with non-Tg mice. Systemic administration of 3-NP resulted in production of hydroxyl free radicals as assessed by the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid. This production was attenuated significantly in Tg-SOD mice. In a similar way, 3-NP produced significant increases in 3-nitrotyrosine/tyrosine, a marker for peroxynitrite-mediated damage, which were significantly attenuated in Tg-SOD mice. These results support that oxygen free radicals and peroxynitrite play an important role in the pathogenesis of 3-NP neurotoxicity.

Notes: Abstract: Metabolic tolerance of low intracellular pH (pHi) was studied in well-oxygenated, perfused, neonatal, rat cerebrocortical brain slices (350 mum thick) by inducing severe hypercapnia. In each of 17 separate experiments 80 brain slices (sim 3.2 g wet weight) were suspended in an NMR tube, perfused with artificial CSF (ACSF), and studied at 4.7 T with 31P and 1H NMR spectroscopy. Spectra obtained every 5 min monitored relative concentrations of lactate or high-energy phosphate metabolites, from which pHi and extracellular pH were determined. Unperturbed slice preparations were metabolically stable for 〉 10 h, with no significant changes occurring in pHi, ATP, phosphocreatine (PCr), inorganic phosphate, or lactate. Different levels of hypercapnia were produced by sequentially perfusing slices with the following different ACSF batches, each having previously been equilibrated with a specific mixture of CO2 in oxygen: (a) 10% CO2, 15 min of perfusion; (b) 30% CO2, 15 min of perfusion; (c) 50% CO2, 15 min of perfusion; (d) 70% CO2, 30 min of perfusion; (e) 50% CO2, 15 min of perfusion; (f) 30% CO2, 15 min of perfusion; and (g) 10% CO2, 15 min of perfusion. At the completion of this protocol slices were again perfused with fresh ACSF that was equilibrated with a 95% O2/5% CO2 gas mixture. In each of five separate 1H and 31P experiments, brain slices were recovered within 2 h after termination of exposure to high CO2. The pHi was determined from measurements of the chemical shift difference between phosphoethanolamine and PCr, using a calibration curve obtained for our preparation. During hypercapnia, pHi decreased from 7.18 pmn 0.22 to 6.79 pmn 0.13, 6.60 pmn 0.11, 6.33 pmn 0.40, and 6.37 pmn 0.14 when CO2 concentrations were increased to 10, 30, 50, and 70%, respectively. Lactate/N-acetylaspartate ratios were unchanged until the administration of 70% CO2, which caused a relative increase from 0.83 pmn 0.15 to 1.35 pmn 0.32 (n = 5, p = 0.0024). The administration of 70% CO2 also caused complete depletion of PCr and a 46 pmn 22% (n = 5, p = 0.0024) decrease in ATP content. Metabolite and pH values returned to control values after restoration of normocapnia. The lowest tissue pHi attainable in our protocol using 30% O2/70% CO2 in our brain slice preparation was sim 6.3.

Notes: Abstract: Traumatic spinal cord injury has recently been shown to cause a rapid increase in free fatty acids (FFAs) and lipid degradation in cats. The present studies report a more delayed, time-dependent increase in FFAs and a concomitant decrease in phospholipids following traumatic spinal injury in rats. The largest percentage increases were found for polyunsaturated fatty acids, particularly arachi-donic acid. Associated with these changes were a reduction in the activity of Na+,K+-ATPase and development of spinal cord edema. These findings support the hypothesis that traumatic spinal cord injury leads to delayed, as well as early, hydrolysis of membrane phospholipids, resulting in the liberation of FFAs. Such changes may contribute to secondary spinal cord injury either through direct effects on membranes or through the actions of secondary metabolic products such as the eicosanoids. The latter may cause tissue injury by contributing to the reduction in spinal cord blood flow or through inflammatory responses that follow trauma.

Notes: We studied whether NS-398, a selective cyclo-oxygenase-2 (COX-2) enzyme inhibitor, and piroxicam, an inhibitor of COX-2 and the constitutively expressed COX-1, protect neurones against hypoxia/reoxygenation injury. Rat spinal cord cultures were exposed to hypoxia for 20 h followed by reoxygenation. Hypoxia/reoxygenation increased lactate dehydrogenase (LDH) release, which was inhibited by piroxicam (180–270 µm) and NS-398 (30 µm). Cell counts confirmed the neuroprotection. Western blotting revealed no COX-1 or COX-2 proteins even after hypoxia/reoxygenation. Production of prostaglandin E2 (PGE2), a marker of COX activity, was barely measurable and piroxicam and NS-398 had no effect on the negligible PGE2 production. Hypoxia/reoxygenation increased nuclear factor-kappa B (NF-κB) binding activity, which was inhibited by piroxicam but not by NS-398. AP-1 binding activity after hypoxia/reoxygenation was inhibited by piroxicam but strongly enhanced by NS-398. However, both COX inhibitors induced activation of extracellular signal-regulated kinase (ERK) in neurones and phosphorylation of heavy molecular weight neurofilaments, cytoskeletal substrates of ERK. It is concluded that piroxicam and NS-398 protect neurones against hypoxia/reperfusion. The protection is independent of COX activity and not solely explained by modulation of NF-κB and AP-1 binding activity. Instead, piroxicam and NS-398-induced phosphorylation through ERK pathway may contribute to the increased neuronal survival.

Notes: Mild insults to neurons caused by ischemia or glutamate induce apoptosis, whereas severe insults induce non apoptotic death, such as necrosis. The molecular targets that are damaged by these insults and ultimately induce cell death are not fully established. To determine if DNA damage can induce apoptotic or non apoptotic death depending on the severity, neurons were treated with up to 128 Gy of ionizing radiation. Such treatment induced a dose-related increase in DNA single-strand breaks but no immediate membrane disruption or lipid peroxidation. Following moderate doses of 〈inlineGraphic alt="leqslant R: less-than-or-eq, slant" extraInfo="nonStandardEntity" href="urn:x-wiley:00223042:JNC0070:les" location="les.gif"/〉 32 Gy, neuronal death had many characteristics of apoptosis including nuclear fragmentation and DNA laddering. Nuclear fragmentation and membrane breakdown after moderate DNA damage could be blocked by inhibition of active protein synthesis with cycloheximide and by inhibition of caspases. In contrast, cell death after doses of 〉 32 Gy was not blocked by cycloheximide or caspase inhibitors, and membrane breakdown occurred relatively early in the cell death process. These data suggest that cell death after high dose irradiation and severe DNA damage can occur by non apoptotic mechanisms and that blocking apoptotic pathways may not prevent death after severe damage.

Notes: Cellular injury was evaluated in superfused cerebrocortical slices (350 µm) from 7-day-old Sprague–Dawley rats exposed to 30 min hypoxia followed by 4 h of reoxygenation. At the end of hypoxia homogenous cytosolic immunoreactivity of cytochrome c increased approximately fourfold, cytochrome c intensity in western blot analyses increased more than fivefold, and whole cell and cytosolic cleaved caspase-9 underwent 50% and 100% increases, respectively. Immunostaining of sections taken 1.5 h after hypoxia showed: (i) more than a threefold increase in cleaved caspase-9; (ii) localization of cleaved caspase-9 to the interior and peripheral exterior of nuclei; and (iii) homogeneously distributed cytochrome c in the cytosol. Western blot analysis for 1.5 h after hypoxia showed that cytosolic caspase-9 returned to control values, while whole cell caspase-9 stayed approximately the same, suggesting translocation of caspase-9 to nuclei. By 4 h after hypoxia there was significant nuclear fragmentation and an increase in TUNEL positive staining. 31P/1H nuclear magnetic resonance (NMR) confirmed substantial decreases of ATP and phosphocreatine during hypoxia, with rapid but incomplete recovery being close to steady state 1 h after reoxygenation. At all time points after hypoxia the primary injury was cytochrome c associated apoptosis.