To confirm the differentiation of DPSCs to a neuronal phenotype and to demonstrate that this differentiation was not an artefact, three analyses were performed: morphological changes, expression of MAP2
, and activation of neuronal markers.
EPA also caused NeuroD and Map2
mRNA levels to increase in Tuj-1-positive cells.
Representative immunofluorescent photographs of astrocyte markers GFAP and MAP2
. Hoechst 33258 used as nuclei staining.
QRT-PCR Analysis of MAP2
, NF-[kappa]B, and NeuN Expression.
Recently, the electromagnetic waves have been demonstrated to modulate the functions of the cytoskeleton and to promote the neuronal differentiation and the neurogenesis in the hippocampus in vivo through the upregulation of the Cav-1 channel, [beta]-III-tubulin, MAP2
, and the brain-derived neurotrophic factor (BDNF) .
In pre-SCI rats, the (350 mg/kg/day) RGE-treated and vehicle-treated groups showed similar patterns of MAP2
immunoreactivity in the spinal cord (Figures 3(a) and 3(b)).
The membranes were blocked by emersion in 5% skim milk on a shaker at room temperature for 1h and then incubated overnight with primary antibodies against GAPDH, [beta]-tubulin III, MAP2
, GFAP, tau, pSer9-GSK-3[beta] (Cell Signaling Technology), and [beta]-catenin.
For quantitative assessment of cell differentiation in control and Paroxetine treated cells, the relative numbers of cells expressing different markers like mature neurons (MAP2
), astrocytes (GFAP), and precursor neuronal cells (Nestin) were counted as percentages of the total DAPI-stained cell population.
was detected 72 h after the treatment under immunofluorescence microscope (Olympus, Japan) at 20x objective as described previously (Gaikwad and Agrawal-Rajput, 2015) using specific mouse monoclonal anti-MAP2 (1:1000) and Secondary antibody (goat anti-mouse IgG/IgM Alexaflour 488/543 (1:5000) (Invitrogen).
Axonal and tissue preservation by immunohistochemistry (i.e., NeuN counts or fluorogold tract tracing), protein quantification (i.e., NF200, MAP2
, CNPase western blot analysis), and cavity formation/tissue preservation imaging (i.e., luxol fast blue-hematoxylin eosin stain) are recommended.
(91,92) Considering that among the proteins phosphorylated by PKA are the CREB transcription factor, (93,94) which stimulates BDNF expression and of its TrkB (49) receptor and MAP2
protein which stimulates the depolymerization of microtubules, (89,92) it is proposed that part of the therapeutic effect is due to the stimulation of the aforementioned cellular and synaptic plasticity processes.