Cell and Tissue Journal

Abstract -
Aim: In this study Nurr1 and GDNF, due to their anti-inflammatory and regenerative effects and Nurr1-mediated regulation of GDNF receptor (Ret) expression, were selected to protect dopaminergic SH-SY5Y cell line against neuroinflammation and toxicity caused by 6-OHDA.
Material and Methods: Recombinant lentiviral vectors carrying Nurr1 and GDNF genes were prepared and transdused to SH-SY5Y and astrocytoma (1321N1) cell lines respectively. Also HEK-293T cells were transfected with plasmid carrying GDNF to overexpress this factor; condition media of transduced astrocytoma and transfected HEK-293T cells were collected and stored. Next, overexpression of mentioned factors was demonstrated by RT-PCR. On the other hand, microglial cells were isolated from neonatal rat brains and induced with LPS to produce neuroinflammatory factors; Inducible expression of them was demonstrated by Griess test and RT-PCR. Condition media of microglia was collected and saved. Finally, SH-SY5Y cells overexpressing Nurr1 were treated with condition media of transduced astrocytoma / transfected HEK-293T and then with condition media of LPS-induced microglia or 6-OHDA toxin.
Results: data from MTT assay showed, SH-SY5Y cells overexpressing Nurr1 or pretreated with GDNF are more resistant to toxicity caused by neuroinflammation and 6-OHDA. Also Nurr1 and GDNF have cooperative effects and give more protection to dopaminergic cells.
Conclusion: Nurr1 and GDNF each have protective effects on dopaminergic neural cells against inflammatory factors or 6-OHDA. Also they synergize with each other leading to more protection for dopaminergic neural cells.

Abstract Aim: The aim of the current study was to isolate microglial cells from neonatal rat brain, activate the cells using lipopolysaccharide (LPS) and examine the effect of the inflammatory factors that they express on dopaminergic (DAergic) neurons.
Materials and Methods: mixed glial cells were isolated from 1-3 day rat neonatal brains and then microglial cells were extracted from them. Upon treatment of the cells with LPS, their conditioned media (CM) were collected. DAergic SH-SY5Y cell line was seeded in 96-well plates and fed with the collected CM. The rate of viability and apoptosis of the neuronal cells was examined using MTT and apoptosis tests and the data were analyzed statistically.
Results: Ten to thirteen days after isolation, mixed glial cells were composed of three types: astroglia, oligodendrocytes and microglia. Microglia were floating while semi-attached to the surface. Twenty four hours post-isolation, these cells were transferred to and cultured in separate dishes where they formed spindled and ramified phenotypes. At this stage, the cells were treated with LPS to be activated. This activation was demonstrated by morphological changes from spindle-like form to amoeboid form, detection of increase in NO expression using Griess test and induction of inflammatory iNOS and TNF-α gene expression. Also teatment of SH-SY5Y cell line with conditioned medium of activated microglia led to both apoptotic and necrotic cell death.
Conclusion: LPS-mediated activation of microglia results in overexpression of neuroinflammatory markers. The overproduction of these markers results in both apoptotic and necrotic cell death amongst DAergic neurons via neuroinflammation.