Research ArticleOriginal Article
Open Access

Neuroprotective role of thymoquinone against 1-methyl-4-phenylpyridinium-induced dopaminergic cell death in primary mesencephalic cell culture

Khaled S. Radad, Mubarak M. Al-Shraim, Mahmoud F. Moustafa and Wolf-Dieter Rausch
Neurosciences Journal January 2015, 20 (1) 10-16;

Article Figures & Data

Figures

  • Figure 1
    Figure 1

    Anti-TH immunohistochemical staining of cultured cells showing: A) Survival of dopaminergic neurons in primary mesencephalic cell cultures. 100% corresponds to the total number of THir neurons after 12 DIV in untreated controls. Values represent the mean±SEM of 3 independent experiments with 4 wells in each treatment. In each well, 10 randomly selected fields were counted for TH immunocytochemistry (#p=0.001, *p=0.008, +p=0.009). B) Representative micrographs of THir neurons after 12 DIV. Untreated control cultures showed THir neurons with long and branched processes (arrows). The MPP+-treated cultures showed THir neurons with few, shortened and thickened neuritis (arrows). Treatment with TQ improves the morphology of THir neurons compared to MPP+-treated cultures. TH - tryosine hydrolase, THir - tyrosine hydroxylase immunoreactive, DIV - day in vitro, SEM - standard error of mean, MPP+ - 1-methyl-4-phenylpyridinium, TQ - thymoquinone

  • Figure 2
    Figure 2

    Lactate dehydrogenase (LDH) release in primary mesencephalic cell cultures. 100% corresponds to LDH activity in the culture medium after 12 DIV. Values represent the mean±SEM of 3 independent experiments with 4 wells in each treatment. (#p=0.0001, *p<0.021, +p=0.012). DIV - day in vitro, SEM - standard error of mean, MPP+ - 1-methyl-4-phenylpyridinium, TQ - thymoquinone

  • Figure 3
    Figure 3

    LysoTracker® Deep Red fluorescence staining of cultured cells showing: A) LysoTracker® Deep Red fluorescence intensity in primary mesencephalic cell cultures. 100% corresponds to the density of LysoTracker® Deep Red in primary mesencephalic cell cultures after 12 DIV. Values represent the mean±SEM of 3 independent experiments with 4 wells in each treatment. Fluorescence intensity was determined densitometrically from 12 randomly selected micrographs in each experiment (3 photos from each well). (#p=0.05, *p=0.0001) B) Representative micrographs showing that treatment of cultures with TQ increased LysoTracker® Deep Red fluorescence intensity compared with MPP+-treated cultures. DIV - day in vitro, SEM - standard error of mean, TQ - thymoquinone, MPP+ - 1-methyl-4-phenylpyridinium

  • Figure 4
    Figure 4

    5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine (JC-1) fluorescence staining of cultured cells showing: A) Red:green fluorescence ratio of JC-1 in primary mesencephalic cell cultures. 100% corresponds to the red:green fluorescence ratio of JC-1 in primary mesencephalic cell cultures after 12 DIV. Values represent the mean±SEM of 3 independent experiments with 4 wells in each treatment. Red:green fluorescence ratio of JC-1 was determined densitometrically from 12 randomly selected micrographs in each experiment (3 photos from each well). (#p=0.0001, *p=0.0001) B) Representative micrographs showing that treatment of cultures with TQ increased red fluorescence compared to MPP+-treated cultures which exhibits marked green fluorescence. DIV - day in vitro, SEM - standard error of mean, TQ - thymoquinone, MPP+ - 1-methyl-4-phenylpyridinium

  • Figure 5
    Figure 5

    4’,6-diamidino-2-phenylindole fluorescence staining of cultured cells showing: A) Number of nuclei showing apoptotic features with condensed and fragmented chromatin in primary mesencephalic cell cultures. 100% corresponds to the number of apoptotic nuclei in untreated control cultures after 12 DIV. Values represent the mean±SEM of 3 independent experiments with 4 wells in each treatment. Twelve photos were taken from each experiment (3 photos from each well). (#p=0.0001, *p=0.0001) B) Representative micrographs showing that treatment of cultures with TQ decreased the number of apoptotic nuclei compared to MPP+-treated cultures. Insert shows apoptotic nuclei (arrow) at 20× magnification. DIV - day in vitro, SEM - standard error of mean, TQ - thymoquinone, MPP+ - 1-methyl-4-phenylpyridinium

Tables

  • Table 1

    Summary of the neuroprotective effect of TQ against MPP+ treatment in primary mesencephalic cell culture.

    Studied parametersUntreated controlsMPP+(10 μM)TQ + MPP+(10 μM)
    0.01 μM0.1 μM1 μM10 μM
    %SEM%SEM%SEM%SEM%SEM%SEM
    THir neurons1003.0060.80#4.5066.833.0169.64*3.3971.24+2.8867.993.17
    LDH release10013.50245.99#30.76--175.40*16.95169.52+16.22--
    LysoTracker® Deep Redintensity1008.75222.52#16.51----682.90*73.92--
    JC-1 red/green fluorescence ratio1002.0883.92#3.16----107.18*2.99--
    DAPI-stained apoptotic nuclei10010.34239.77#12.88----139.20*8.81--
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