Redistribution of Glutamate and Glutamine in Slices of Human Neocortex Exposed to Combined Hypoxia and Glucose Deprivation in vitro

Abstract

Summary: This study was undertaken to elucidate the roles of neurons and glial cells in the handling of gluta mate and glutamine, a glutamate precursor, during cere bral ischemia. Slices (400--6 00 fLm) from human neocortex obtained during surgery for epilepsy or brain tumors were incubated in artificial cerebrospinal fluid and subjected to 30 min of combined hypoxia and glucose deprivation (an in vitro model of brain ischemia). These slices, and con trol slices that had not been subjected to "ischemic" con ditions, were then fixed and embedded. Ultrathin sec tions were processed according to a postembedding im munocytochemical method with polyclonal antibodies raised against glutamate or glutamine, followed by colloi dal gold-labeled secondary antibodies. The gold particle densities over various tissue profiles were calculated from electron micrographs using a specially designed computer program. Combined hypoxia and glucose dep rivation caused a reduced glutamate immunolabeling in neuronal somata, while that of glial processes increased. Following 1 h of recovery, the glutamate labeling of neu ronal somata declined further to very low values, com pared to control slices. The glutamate labeling of glial cells returned to normal levels following recovery. InThe pathogenesis of ischemic brain damage is not fully understood, and a number of neurotoxic sub stances has been implicated (Krause et aI. , 1988; Siesjo, 1988;Ginsberg, 1990; Siesjo et aI. , 1990 503axon terminals, no consistent change in the level of glu tamate immunolabeling was observed. Immunolabeling of glutamine was low in both nerve terminals and neuronal somata in normal slices and was reduced to nondetectable levels in nerve terminals upon hypoxia and glucose dep rivation. This treatment was also associated with a re duced glutamine immunolabeling in glial cells. Reversed glutamate uptake due to perturbations of the transmem brane ion concentrations and membrane potential proba bly contributes to the loss of neuronal glutamate under "ischemic" conditions. The increased glutamate labeling of glial cells under the same conditions can best be ex plained by assuming that glial cells resist a reversal of glutamate uptake, and that their ability to convert gluta mate into glutamine is compromised due to the energy failure. The persistence of a nerve terminal pool of glu tamate is compatible with recent biochemical data indi cating that the exocytotic glutamate release is contingent on an adequate energy supply and therefore impeded dur ing ischemia.

Perinatal Iron Deficiency Predisposes the Developing Rat Hippocampus to Greater Injury from Mild to Moderate Hypoxia—Ischemia

“…Typically, glutamate released into the extracellular space is taken up by nearby astrocytes, converted to glutamine through the action of the enzyme glutamine synthase and trafficked back to the neurons for reconversion to glutamate, thereby completing the so-called glutamate-glutamine cycle (Daikhin and Yudkoff, 2000;Magistretti et al 1999). The decreased glutamate and increased glutamine concentrations in the present study implies that after acute HI injury, the neuronal-glial glutamate-glutamine equilibrium may be shifted in favor of increased glutamine synthesis in the iron-deficient hippocampus (Aas et al, 1993;Dao et al, 1991;Wallin et al, 2000;Phillis and O'Regan, 2003). Such metabolic shift has been showed in in vivo rat models of glutamate receptor overstimulation (Tkac et al, 2001).…”

Hyperoxia promotes astrocyte cell death after oxygen and glucose deprivation

“…Astrocytes are integrally involved in metabolic and ionic homeostasis, inflammatory responses, and control of extracellular glutamate levels (Aas et al, 1993;Hertz and Zielke, 2004;Hertz et al, 1999;Storm-Mathisen et al, 1992;Torp et al, 1994). Astrocytes also produce neurotrophic factors that promote neuronal survival and provide neurons with precursors for glutathione biosyn-thesis, which is necessary to combat oxidative stress (Dringen, 2000;Drukarch et al, 1997;Makarov et al, 2002).…”

Metabolic changes in quinolinic acid‐lesioned rat striatum detected non‐invasively by in vivo 1H NMR spectroscopy

“…The changes were highly significant for both Glu and Gln, however, the change in the sum of both was 0.3 Ϯ 0.6 mol/g, which was not significant (P Ͼ 0.6). Although the metabolite changes could be perceived as reflecting a shift in cellular composition of the tissue examined, the constancy of ([Glu] ϩ [Gln]) may also imply a metabolic shift similar to what has been observed in ischemia, namely an increased Gln concentration (in astrocytes) and a decreased (neuronal) glutamate concentration (Aas et al, 1993). Such a metabolic blockade at the level of the conversion of glutamine to glutamate (or glutamine transport out of the astrocyte) would imply that the action of QA on metabolism is indirect, by affecting the glutamine-glutamate interconversion, which has been linked to glutamatergic action (Yudkoff et al, 1993;Gruetter et al, 1998a;Sibson et al, 1998).…”