Transgene expression of plasmid DNAs directed by viral or neural promoters in the rat brain.

The use of circular plasmid DNA may be an alternative method for the transfer of genes into the brain and is presumably easier to use than other vectors, such as viruses or genetically engineered cells. The effectiveness and time course of the expression of a reporter gene (LacZ), directed by appropriate promoters, was studied after stereotaxic injection of naked plasmid DNAs into the rat thalamus, cortex or cerebellum. The efficiencies of three different promoters, the human cytomegalovirus (HCMV) promoter and the glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) promoters (specific for astrocytes and neurons, respectively) to drive reporter gene expression were compared. Efficient expression of beta-gal, detected by X-gal histochemistry or immunochemistry, required the use of 50 microg of DNA and was detectable as early as 48 h after injection. Expression increased until day 8, remained stable until day 15, then decreased over 2 months, probably as a result of non-specific degradation of the plasmids within the transfected cells rather than from specific down-regulation of promoters, as the same time course was seen with all three promoters tested. Depending on the promoter used (GFAP or NSE), LacZ was preferentially expressed within astrocytes or neurons, respectively. The GFAP promoter was found to be as efficient as the HCMV promoter, possibly due to the reactive gliosis induced by plasmid injection which is known to up-regulate GFAP expression.

Glutamate-modulated production of GABA in immortalized astrocytes transduced by a glutamic acid decarboxylase-expressing retrovirus.

Replication-defective Moloney murine leukemia virus expressing the GAD67 gene under the control of the GFAP promoter was produced using selected clones of a fibroblast-packaging cell line. A spontaneously immortalized astrocyte cell line was infected with this virus and cellular clones expressing GAD67 selected. Astrocyte and fibroblast clones expressed functional GAD (detected by glutamic acid decarboxylation), but only fibroblasts were able to also produce GABA in the extracellular medium. When exposed to 200 microM glutamate, despite an observed difference in the rates of glutamate accumulation in control and GAD67-expressing astrocytes, similar proportions of glutamate taken up were detected. In GAD67-expressing astrocytes, the glutamate was mainly converted into GABA, suggesting GAD transgene activity to be dominant over other glutamate metabolic pathways, such as glutamine synthetase and glutamate dehydrogenase. Moreover, rapid GABA release into the cell medium was also observed, suggesting the involvement of reverse GABA transporters. The use of the GFAP promoter might be able to take advantage of its activation in response to factors inducing reactive gliosis observed in pathological insults. GAD67-expressing astrocytes might therefore be used for future grafting in pathological situations in which an excess of glutamate results in neuronal dysfunction or cell death.

Enhanced neuronal protection from oxidative stress by coculture with glutamic acid decarboxylase-expressing astrocytes.

Astrocytes expressing glutamic acid decarboxylase GAD67 directed by the glial fibrillary acidic protein promoter were shown to provide enhanced protection of PC12 cells from H(2)O(2) treatment and serum deprivation in the presence of glutamate. In addition, they protected non-differentiated, but not differentiated, embryonic rat cortical neurons from glutamate toxicity. Glutamic acid decarboxylase (GAD)-expressing astrocytes showed increased glutathione synthesis and release compared to control astrocytes. These changes were due to GAD transgene expression, as transient expression of a GAD antisense plasmid resulted in partial suppression of the increase in glutathione release. In addition to the previously demonstrated increases in NADH and ATP levels and lactate release, GAD-expressing astrocytes show increased antioxidant activity, explaining their ability to protect neurons from various injuries.

Glutamic acid decarboxylase-expressing astrocytes exhibit enhanced energetic metabolism and increase PC12 cell survival under glucose deprivation.

Astrocytes play a key role by catabolizing glutamate from extracellular space into glutamine and tricarboxylic acid components. We previously produced an astrocytic cell line that constitutively expressed glutamic acid decarboxylase (GAD67), which converts glutamate into GABA to increase the capacity of astrocytes to metabolize glutamate. In this study, GAD-expressing astrocytes in the presence of glutamate were shown to have increased energy metabolism, as determined by a moderate increase of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, by an increased ATP level, and by enhanced lactate release. These changes were due to GAD transgene expression because transient expression of a GAD antisense plasmid resulted in partial suppression of the ATP level increase. These astrocytes had an increased survival in response to glucose deprivation in the presence of glutamate compared with the parental astrocytes, and they were also able to enhance survival of a neuronal-like cell line (PC12) under glucose deprivation. This protection may be partially due to the increased lactate release by GAD-expressing astrocytes because PC12 cell survival was enhanced by lactate and pyruvate under glucose deprivation. These results suggest that the establishment of GAD expression in astrocytes enhancing glutamate catabolism could be an interesting strategy to increase neuronal survival under hypoglycemia conditions.

Monitoring gamma-aminobutyric acid in human brain and plasma microdialysates using micellar electrokinetic chromatography and laser-induced fluorescence detection.

Due to its low electrophoretic mobility, few studies have been able to measure gamma aminobutyric acid (GABA) in biological samples by means of capillary zone electrophoresis. Nevertheless, in micellar electrokinetic chromatography (MEKC) by adding a surfactant to the mobile phase separation can be carried out on the basis of the partition coefficient of the molecules rather than their electrophoretic mobility. In the present study microdialysis coupled to MEKC with laser induced fluorescence detection was used to successfully monitor GABA from cerebrospinal fluid and plasma dialysates. Moreover, we monitored changes in extracellular GABA from a human brain. Microdialysis samples were collected from a Parkinson's disease patient undergoing a thallamotomy as part of her treatment. Significant decreases in extracellular GABA were detected during high frequency electrical stimulation and following a thermolesion of the thalamus. These results demonstrate the feasibility of MEKC coupled to laser-induced fluorescence detection in resolving neutral amino acids, specifically GABA, from different human body fluids.

Microdialisis cerebral en humanos: experiencia en la enfermedad de parkinson / Brain microdialysis in human: experienci in the Parkinson disease

Se ha efectuado el procedimiento de microdiálisis cerebral durante talamotomía estereotáxica N.V.L en 3 pacientes de enfermedad de parkinson, para corregirles el síndrome del temblor. Se realizó correlación entre la concentración intersticial del GABA y los cambios de intensidad del tremor. La microdiálisis es un método seguro, reproductible y fácil de practicar durante operaciones neuroquirúrgicas estereotáxicas. Se expone los resultados preliminares. Debido a la muestra de 3 pacientes, no se puede establecer conclusiones definitivas. Esta investigación está actualmente en progreso

Braquiterapia estereotáxica cerebral en Venezuela: nota histórica / Cerebral stereotactic brachytherapy in Venezuela: historical note

La braquiterapia o radioterapia intersticial cerebral inicia con Leksell en 1951. Ha evolucionado con los aportes de Backlund y su amplio estudio del efecto del p32 en craneofaringiomas quísticos, complementándose esta modalidad terapéutica con radio-gamma-cirugía cuando el componente sólido de la lesión así lo ameritaba. La experiencia es transmitida a través de algunos de los autores (JFDC y RG) a Venezuela donde se continuó y donde se siguen aportando modificaciones, con la utilización de otro radioisótopo, el I131 y ampliando su aplicación a otros tipos de tumores quísticos como los de la región pineal