Notes: Abstract: We have recently shown that brain slices are capable of metabolizing arachidonic acid by the epoxy-genase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types to form epoxygenase metabolites. We have examined the astrocyte epoxygenase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte homogenate, when incubated with radiolabeled [3H]-arachidonic acid, formed products that eluted in four major groups designated as R17–30, R42–50, R51–82, and R83–90 based on their retention times in reverse-phase HPLC. These fractions were further segregated into as many as 13 peaks by normal-phase HPLC and a second reverse-phase HPLC system. The principal components in each peak were structurally characterized by gas chromatography/electron impact-mass spectrometry. Based on HPLC retention times and gas chromatography/electron impactmass spectrometry analysis, the more polar fractions (R17–30) contained prostaglandin D2 as the major cyclooxygenase product. Minor products included 6-keto prostaglandin F1α, prostaglandin E2, prostaglandin F2α, and thromboxane B2. Fractions R42–50, R51–82. and R83–90 contained epoxygenase and lipoxygenase-like products. The major metabolite in fractions R83–90 was 5, 6-epoxyeicosatrienoic acid (EET). Fractions R51–82 contained 14, 15-and 8, 9-EETs, 12-and 5-hydroxyeicosatetraenoic acids, and 8, 9-and 5, 6-dihydroxyeicosatrienoic acids (DHETs). In fractions R42–50, 14, 15-DHET was the major product. When radiolabeled [3H]14, 15-EET was incubated with astrocyte homogenate, it was rapidly metabolized to [3H]14, 15-DHET. The metabolism was inhibited by submicromolar concentration of 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase activity. Formation of other polar metabolites such as triols or epoxyalcohols from 14, 15-DHET was not observed. In conclusion, astro-cytes readily metabolize arachidonic acid to 14, 15-EET, 5, 6-EET, and their vicinal-diols. Previous studies suggest these products may affect neuronal function and cerebral blood flow.

Notes: Abstract: Our previous studies have shown that 14,15-epoxyeicosatrienoic acid (14,15-EET) is a major product of arachidonic acid metabolism in astrocytes. The purpose of this study was to investigate cellular regulation of 14,15-EET incorporation, distribution, and metabolism in primary cultures of rat brain cortical astrocytes. Incorporation of 14,15-EET into astrocytes was lower (93,390 ± 11,121 dpm/5 × 106 cells) than incorporation of 8,9-EET (226,500 ± 5,567 dpm/5 × 106 cells) and arachidonic acid (321,600 ± 1,200 dpm/5 × 106 cells). 14,15-EET was distributed in the order neutral lipids and free fatty acids (solvent front) ≫ phosphatidylcholine (PC) 〉 phosphatidylinositol (PI) 〉 phosphatidylethanolamine. In contrast, 8,9-EET and arachidonic acid were exclusively incorporated into PC. During incubation, astroglial epoxide hydrolase selectively metabolized 14,15-EET, but not 8,9-EET, to its vic-diol. Although 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase, completely inhibited 14,15-EET metabolism, a large amount of cell-incorporated radioactivity remained as free 14,15-EET. Long-term exposure of astrocytes to 4β-phorbol 12-myristate 13-acetate (4β-PMA) resulted in a time-dependent incorporation of 14,15-EET into PI but not in control cells exposed to 4α-phorbol 12,13-didecanoate. PKC down-regulation completely inhibited epoxide hydrolase metabolism of 14,15-EET. Following recovery of down-regulated PKC, 1 week after treatment with 4β-PMA, astrocytes regained their normal pattern of low incorporation of 14,15-EET. Protein kinase C (PKC) inhibition by staurosporine enhanced 14,15-EET incorporation without affecting its metabolism to 14,15-dihydroxyeicosatrienoic acid. Incorporation of 14,15-EET by PKC-down-regulated cells was inhibited by thimerosal, a known inhibitor of fatty acyl-CoA synthase. Our results suggest that the lower incorporation of 14,15-EET into astroglial cells may be due to modulation of PKC-mediated cellular mechanism(s).

Notes: S100B protein in brain is produced primarily by astrocytes, has been used as a marker for brain injury and has also been shown to be neurotrophic and neuroprotective. Using a well characterized in vitro model of brain cell trauma, we examined the potential role of exogenous S100B in preventing delayed neuronal injury. Neuronal plus glial cultures were grown on a deformable Silastic membrane and then subjected to strain (stretch) injury produced by a 50 ms displacement of the membrane. We have previously shown that this injury causes an immediate, but transient, nuclear uptake of the fluorescent dye propidium iodide by astrocytes and a 24–48 h delayed uptake by neurons. Strain injury caused immediate release of S100-beta with further release by 24 and 48 h. Adding 10 or 100 nm S100B to injured cultures at 15 s, 6 h or 24 h after injury reduced delayed neuronal injury measured at 48 h. Exogenous S100B was present in the cultures through 48 h. These studies directly demonstrate the release and neuroprotective role of S100B after traumatic injury and that, unlike most receptor antagonists used for the treatment of trauma, S100B is neuroprotective when given at later, more therapeutically relevant time points.

Notes: Abstract: Energy deficit after traumatic brain injury (TBI) may alter ionic homeostasis, neurotransmission, biosynthesis, and cellular transport. Using an in vitro model for TBI, we tested the hypothesis that stretch-induced injury alters mitochondrial membrane potential (Δ?m) and ATP in astrocytes and neurons. Astrocytes, pure neuronal cultures, and mixed neuronal plus glial cultures grown on Silastic membranes were subjected to mild, moderate, and severe stretch. After injury, Δ?m was measured using rhodamine-123, and ATP was quantified with a luciferin-luciferase assay. In astrocytes, Δ?m dropped significantly, and ATP content declined 43-52% 15 min after mild or moderate stretch but recovered by 24 h. In pure neurons, Δ?m declined at 15 min only in the severely stretched group. At 48 h postinjury, Δ?m remained decreased in severely stretched neurons and dropped in moderately stretched neurons. Intracellular ATP content did not change in any group of injured pure neurons. We also found that astrocytes and neurons release ATP extracellularly following injury. In contrast to pure neurons, Δ?m in neurons of mixed neuronal plus glial cultures declined 15 min after mild, moderate, or severe stretch and recovered by 24-48 h. ATP content in mixed cultures declined 22-28% after mild to severe stretch with recovery by 24 h. Our findings demonstrate that injury causes mitochondrial dysfunction in astrocytes and suggest that astrocyte injury alters mitochondrial function in local neurons.

Notes: Abstract: Current literature suggests that a massive influx of Ca2+ into the cells of the CNS induces cell damage associated with traumatic brain injury (TBI). Using an in vitro model for stretch-induced cell injury developed by our laboratory, we have investigated the role of extracellular Ca2+ in astrocyte injury. The degree of injury was assessed by measurement of propidium iodide uptake and release of lactate dehydrogenase. Based on results of in vivo models of TBI developed by others, our initial hypothesis was that decreasing extracellular Ca2+ would result in a reduction in astrocyte injury. Quite unexpectedly, our results indicate that decreasing extracellular Ca2+ to levels observed after in vivo TBI increased astrocyte injury. Elevating the extracellular Ca2+ content to twofold above physiological levels (2 mM) produced a reduction in cell injury. The reduction in injury afforded by Ca2+ could not be mimicked with Ba2+, Mn2+, Zn2+, or Mg2+, suggesting that a Ca2+-specific mechanism is involved. Using 45Ca2+, we demonstrate that injury induces a rapid influx of extracellular Ca2+ into the astrocyte, achieving an elevation in total cell-associated Ca2+ content two- to threefold above basal levels. Pharmacological elevation of intracellular Ca2+ levels with the Ca2+ ionophore A23187 or thapsigargin before injury dramatically reduced astrocyte injury. Our data suggest that, contrary to popular assumptions, an elevation of total cell-associated Ca2+ reduces astrocyte injury produced by a traumatic insult.