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Background Neurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer’s disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor β (hNGFβ) gene expression mediated by recombinant adeno-associated viruses type-2 (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption.Methods rAAV-2 containing hNGFβ gene was constructed. The ability of hNGFβ gene mediated by rAAV-2 vector (rAAV-2/hNGFβ) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFβ in the culture supernatant of BHK-21 cells infected by rAAV-2/hNGFβ. rAAV-2/hNGFβ and rAAV-2/green fluorescence protein (GFP) were administrated separately to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hNGFβ concentration was measured by ELISA and GFP in brain sections was examined by laser scan confocal microscope.Results After 48 hours, hNGFβ content in supernatant was up to (188.0±28.6) pg/ml when BHK-21 cells were infected by rAAV-2/hNGFβ at multiplicity of infection (MOI)1.0×106 vector genome. Neurone fibre outgrowths were obvious in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2/hNGFβ. Whole brain hNGFβ content in rAAV-2/hNGFβ transferred group was up to (636.2±140.6) pg/ml. hNGFβ content of BBB disruption in rAAV-2/hNGFβ infused group increased significantly compared to the control group (P<0.05). GFP expression was clearly observed in brain sections of rAAV-2/GFP transferred group.Conclusion rAAV-2/hNGFβ successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol.
Background Neurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer’s disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor β (hNGFβ) gene expression mediated by recombinant adeno-associated (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption. Methods rAAV-2 containing hNGFβ gene was constructed. The ability of hNGFβ gene mediated by rAAV- -2 / hNGFβ) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFβ in the culture supernatant of BHK-21 cells infected by rAAV-2 / hNGFβ. RAAV-2 / hNGFβ and rAAV- 2 / green fluorescence protein GFP) were administrated to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hNGFβ concentration was measured by ELISA and GFP in brain sections was examined d by laser scan confocal microscope. Results After 48 hours, hNGFβ content in supernatant was up to (188.0 ± 28.6) pg / ml when BHK-21 cells were infected by rAAV-2 / hNGFβ at multiplicity of infection (MOI) vector genome. Neurone fiber outgrowths were significantly in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2 / hNGF [beta]. Whole brain hNGFβ content in rAAV-2 / hNGFβ transferred group was up to ( 636.2 ± 140.6) pg / ml hNGFβ content of BBB disruption in rAAV-2 / hNGFβ infused group increased significantly compared to the control group (P <0.05). GFP expression was clearly observed in brain sections of rAAV-2 / GFP transferred group .Conclusion rAAV-2 / hNGFβ successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol.