RESUMO
OBJECTIVE: Integrin binding to extracellular matrix ligands, including those presenting RGD motifs, modulate diverse cellular processes. In the brain, many endogenous RGD-containing molecules are induced after damage. Previously, the gene therapy vector termed NLSCt, which displays an RGD motif, was shown to neuroprotect after immature brain excitotoxicity. We analyze whether neuroprotection is mediated by the RGD motif. METHODS: RGD-containing synthetic peptide GPenGRGDSPCA (GPen) was injected 2 hours after N-methyl-D-aspartate-mediated excitotoxicity to the postnatal day 9 rat brain. Damage and glial/inflammatory response were evaluated 3 days later. In addition, the neuroprotective effect of GPen and NLSCt after N-methyl-D-aspartate-induced cell death was also analyzed in vitro using neuron-purified and mixed neuron-glia primary cultures. To further characterize whether the neuroprotective effect was mediated by glial-derived soluble factors, we also tested the protective ability of conditioned media from RGD-treated microglia, astrocyte, or mixed glia cultures. RESULTS: Animals treated with GPen peptide showed functional improvement, a significant reduction in lesion volume up to 28%, and a decrease in the number of degenerating neurons. In addition, N-methyl-D-aspartate-injected animals treated with both RGD-containing molecules at the neuroprotective doses showed a significant increase in microglial reactivity and microglia/macrophage cell number, but no differences in neutrophil infiltration and the astroglial response. Finally, in vitro studies showed that the neuroprotective effect was observed in mixed neuron-glia, but not in neuron-purified cultures. Conditioned media from RGD-treated microglial, astroglial, and mixed-glial cultures were not protective. INTERPRETATION: These results suggest that RGD-containing molecules neuroprotect by a glial-dependent mechanism.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Aminoácidos Excitatórios/toxicidade , N-Metilaspartato/toxicidade , Neuroglia/fisiologia , Fármacos Neuroprotetores , Síndromes Neurotóxicas/prevenção & controle , Oligopeptídeos/uso terapêutico , Animais , Astrócitos/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Interpretação Estatística de Dados , Densitometria , Feminino , Imuno-Histoquímica , Microglia/efeitos dos fármacos , Degeneração Neural/prevenção & controle , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Síndromes Neurotóxicas/psicologia , Infiltração de Neutrófilos/efeitos dos fármacos , Gravidez , Ratos , Ratos Long-EvansRESUMO
BACKGROUND: Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn SOD or the control GFP transgenes 2 hours after intracortical N-methyl-D-aspartate (NMDA) administration, and daily functional evaluation was performed. Moreover, 3 days after, lesion volume, neuronal degeneration and nitrotyrosine immunoreactivity were evaluated. RESULTS: Overexpression of Cu/Zn SOD transgene after NMDA administration showed improved functional outcome and a reduced lesion volume at 3 days post lesion. In secondary degenerative areas, increased neuronal survival as well as decreased numbers of degenerating neurons and nitrotyrosine immunoreactivity was seen. Interestingly, injection of the NLSCt vector carrying the control GFP transgene also displayed a significant neuroprotective effect but less pronounced. CONCLUSION: When the appropriate levels of Cu/Zn SOD are expressed transiently after injury using the non-viral modular protein vector NLSCt a neuroprotective effect is seen. Thus recombinant modular protein vectors may be suitable for in vivo gene therapy, and Cu/Zn SOD should be considered as an interesting therapeutic transgene.