Journal: Frontiers in Molecular Neuroscience

Article Title: Noradrenaline Acting on Alpha1 Adrenoceptor as well as by Chelating Iron Reduces Oxidative Burden on the Brain: Implications With Rapid Eye Movement Sleep

doi: 10.3389/fnmol.2019.00007

Figure Lengend Snippet: Percentage changes in Neuro2a- and C6-cell viability after 12 h and 24 h treatment with different doses of NA and H 2 O 2 are shown. Viability of both Neuro2a (A) and C6 (B) was least affected (<15% of control) by treatment with up to 10 μM of NA, whereas H 2 O 2 affected the cell viability in even much lesser concentration. * p < 0.05, ** p < 0.01, *** p < 0.001 as compared to control. Abbreviations are as in the text. (C) Representative microscopic fields of images of Neuro2a and C6 cells with or without treatment with NA (10 μM), H 2 O 2 or their combination are shown. Both Neuro2a as well as C6 cells showed less adherence to the petri-dish. Those remained adhered, showed membrane granulation and more circular (marked by arrows as representative), which are signs of cellular stress/damage or approaching death upon H 2 O 2 treatment for 24 h. Cells treated with H 2 O 2 in presence of NA showed reduced -detachment, -granulation and -circular shaped in addition to lesser death. Scale bar 100 μm.

Article Snippet: The Neuro2a ( RRID:CVCL_0470 ) and C6 cells ( RRID:CVCL_0194 ) were obtained from the National Centre for Cell Science cell repository (Pune, India).

Techniques: Control, Concentration Assay, Membrane

Journal: Frontiers in Molecular Neuroscience

Article Title: Noradrenaline Acting on Alpha1 Adrenoceptor as well as by Chelating Iron Reduces Oxidative Burden on the Brain: Implications With Rapid Eye Movement Sleep

doi: 10.3389/fnmol.2019.00007

Figure Lengend Snippet: Changes in LP (mean ± SEM nmol of MDA) and intracellular ROS levels (as expressed by DCF fluorescence) in Neuro2a and C6 cells under control and various treatment conditions are shown as box-plots. Confluent cell culture containing about 2 × 10 7 cells grown in 100 mm petri dishes were treated with Vit E, NA (10 μM), PRZ, H 2 O 2 alone or in combinations (as shown in respective bars). Vit E (except in Neuro2a), NA and PRZ alone were by and large ineffective in modulating the LP. H 2 O 2 induced increased LP was reduced by NA and Vit E in both Neuro2a (A) and C6 (B) . The protective effect of NA on H 2 O 2 induced LP was lost in presence of PRZ. As shown in (C,D) in both Neuro2a and C6 respectively, ROS was increased by H 2 O 2 , while it was decreased by NA and Vit E. The H 2 O 2 induced effects were prevented by NA and Vit E. As the NA induced effects were not prevented by AR antagonists, PRZ or PRP, it suggested that the effects were not mediated through (ARs). *** p < 0.001 as compared to untreated control and $$ p < 0.01 in comparison with H 2 O 2 treated cells. N = 4 sets of experiments triplicate readings of ×10 5 cells for ROS. Abbreviation as in the text.

Article Snippet: The Neuro2a ( RRID:CVCL_0470 ) and C6 cells ( RRID:CVCL_0194 ) were obtained from the National Centre for Cell Science cell repository (Pune, India).

Techniques: Fluorescence, Control, Cell Culture, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Noradrenaline Acting on Alpha1 Adrenoceptor as well as by Chelating Iron Reduces Oxidative Burden on the Brain: Implications With Rapid Eye Movement Sleep

doi: 10.3389/fnmol.2019.00007

Figure Lengend Snippet: NA mediated modulation of intracellular ROS levels in presence of iron in Neuro2a (A) and C6 (B) is shown. 2 × 10 5 cells per well were seeded in 96 well plates and incubated for 12 h in serum free medium lacking iron or in medium containing 10 μM FeSO 4 as source of iron (Fe 2+ ). Cells in both the media were treated with H 2 O 2 alone or in presence of NA or DFX for 2 h. At the end the cells were incubated with medium containing 25 μM DCFDA for 30 min followed by PBS washing and DCF fluorescence estimation. In the presence of Fe 2+ , H 2 O 2 enhanced ROS generation in both the cells, Neuro2a (A) and C6 (B) , which were prevented by DFX and NA. ** p < 0.01, *** p < 0.001 as compared with control. Abbreviations are in the text.

Article Snippet: The Neuro2a ( RRID:CVCL_0470 ) and C6 cells ( RRID:CVCL_0194 ) were obtained from the National Centre for Cell Science cell repository (Pune, India).

Techniques: Incubation, Fluorescence, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Noradrenaline Acting on Alpha1 Adrenoceptor as well as by Chelating Iron Reduces Oxidative Burden on the Brain: Implications With Rapid Eye Movement Sleep

doi: 10.3389/fnmol.2019.00007

Figure Lengend Snippet: Protective role of NA (10 μM) on H 2 O 2 -induced ROS generation and cell death in Neuro2a (upper panels, A–D ) and C6 (lower panels, E–H ) is shown. Representative photomicrographs of intracellular DCF fluorescence indicative of ROS levels in Neuro2a (A) and C6 (E) are shown. Cells treated with H 2 O 2 alone showed significantly higher fluorescence per cell, while NA treated cells showed significantly reduced fluorescence per cell upon H 2 O 2 treatment. Corresponding histograms of relative fluorescence intensities per cell (mean of 500–600 cells per treatment) from five sets of experiments of Neuro2a (B) and C6 (F) are shown. In separate sets of experiments, the cells were treated for 24 h and percent dead cells were quantified using trypan blue assay. H 2 O 2 significantly increased cell death, which was protected in presence of NA for Neuro2a (C) and C6 (G) . Also, using DAPI (stains all live and dead cells) and PI (stains only dead cells) the proportion of dead cells were quantified in coverslip-grown cells treated with NA, H 2 O 2 or both together. Significantly more number of nuclei were PI (red) stained by treatment of H 2 O 2 alone as compared to NA alone and NA+H 2 O 2 . Representative photomicrographs (20X) of DAPI and PI stained Neuro2a (D) and C6 (H) are shown. PI stained cell shown in red are dead while DAPI stained all nuclei irrespective of live or dead are shown in blue; PI stained cells are seen more in H 2 O 2 treated groups. All readings were taken in duplicate and three ( N = 3) such sets of experiments were conducted. *** p < 0.001 as compared to untreated cells, while $$ p < 0.01 as compared to H 2 O 2 treated cells. Abbreviations are as in the text.

Article Snippet: The Neuro2a ( RRID:CVCL_0470 ) and C6 cells ( RRID:CVCL_0194 ) were obtained from the National Centre for Cell Science cell repository (Pune, India).

Techniques: Fluorescence, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Noradrenaline Acting on Alpha1 Adrenoceptor as well as by Chelating Iron Reduces Oxidative Burden on the Brain: Implications With Rapid Eye Movement Sleep

doi: 10.3389/fnmol.2019.00007

Figure Lengend Snippet: Dose response of NA on ROS generation in C6 cells is shown. At lower concentration (10 μM) NA reduced ROS levels, while at higher concentration (100 μM) there was significant increase in intracellular ROS and it was damaging. * p < 0.05 and *** p < 0.001 in comparison to untreated controls.

Article Snippet: The Neuro2a ( RRID:CVCL_0470 ) and C6 cells ( RRID:CVCL_0194 ) were obtained from the National Centre for Cell Science cell repository (Pune, India).

Techniques: Concentration Assay, Comparison

Journal: PLoS ONE

Article Title: Water Extract from the Leaves of Withania somnifera Protect RA Differentiated C6 and IMR-32 Cells against Glutamate-Induced Excitotoxicity

doi: 10.1371/journal.pone.0037080

Figure Lengend Snippet: Cell viability and toxicity of various concentrations of glutamate was assayed by MTT and LDH assays in RA differentiated C6 (b,c) and IMR-32 (g,h) cells. (d) and (i) histograms represents the relative percentage viability of glutamate and ASH-WEX treated C6 and IMR-32 cells, respectively, as compared to the control cells. (e) and (j) histograms represents the relative LDH activity when the control and ASH-WEX pretreated cells were exposed to different glutamate concentrations. “*” represents the statistical significant difference between all the treatment groups (glutamate alone or glutamate + ASH-WEX groups) with respect to control group. “#” represents the statistical difference between “glutamate + ASH-WEX” treated groups with their respective “glutamate” treatment groups. “*” and “#” = p<0.05.

Article Snippet: C6 (rat glioma) and IMR-32 (human neuroblastoma) cells were purchased from National Centre for Cell Science (Pune, India).

Techniques: Control, Activity Assay

Journal: PLoS ONE

Article Title: Water Extract from the Leaves of Withania somnifera Protect RA Differentiated C6 and IMR-32 Cells against Glutamate-Induced Excitotoxicity

doi: 10.1371/journal.pone.0037080

Figure Lengend Snippet: RT-PCR results for GFAP and NF200 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry analysis was represented by histograms. The expression of GFAP in C6 (c) and NF200 in IMR-32 (g) cells was analysed by immunocytostaining and relative intensity was plotted as histogram as analysed by Image pro-plus software. “*” represents the statistical significant difference between all the treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. “#” represents the statistical difference between “glutamate + ASH-WEX” treated groups with their respective “glutamate” treatment groups. “*” and “#” = p<0.05.

Article Snippet: C6 (rat glioma) and IMR-32 (human neuroblastoma) cells were purchased from National Centre for Cell Science (Pune, India).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Software, Control

Journal: PLoS ONE

Article Title: Water Extract from the Leaves of Withania somnifera Protect RA Differentiated C6 and IMR-32 Cells against Glutamate-Induced Excitotoxicity

doi: 10.1371/journal.pone.0037080

Figure Lengend Snippet: RT-PCR results for HSP70 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry analysis was represented by histograms. The expression of HSP70 in C6 (c) and IMR-32 (g) cells was analysed by immunocytostaining and relative intensity was plotted as histogram as analysed by Image pro-plus software. “*” represents the statistical significant difference between all the treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. “#” represents the statistical difference between “glutamate + ASH-WEX” treated groups with their respective “glutamate” treatment groups. “*” and “#” = p<0.05.

Article Snippet: C6 (rat glioma) and IMR-32 (human neuroblastoma) cells were purchased from National Centre for Cell Science (Pune, India).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Software, Control

Journal: PLoS ONE

Article Title: Water Extract from the Leaves of Withania somnifera Protect RA Differentiated C6 and IMR-32 Cells against Glutamate-Induced Excitotoxicity

doi: 10.1371/journal.pone.0037080

Figure Lengend Snippet: RT-PCR results for NCAM mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented by histograms. The expression of NCAM in C6 (c) and IMR-32 (f) cells was analysed by immunostaining. “*” represents the statistical significant difference between all the treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. “#” represents the statistical difference between “glutamate + ASH-WEX” treated groups with their respective “glutamate” treatment groups. “*” and “#” = p<0.05.

Article Snippet: C6 (rat glioma) and IMR-32 (human neuroblastoma) cells were purchased from National Centre for Cell Science (Pune, India).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunostaining, Control

Journal: PLoS ONE

Article Title: Water Extract from the Leaves of Withania somnifera Protect RA Differentiated C6 and IMR-32 Cells against Glutamate-Induced Excitotoxicity

doi: 10.1371/journal.pone.0037080

Figure Lengend Snippet: RT-PCR results for PST mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented by histograms. The expression of PSA-NCAM in C6 (c) and IMR-32 (f) cells was analysed by immunostaining. “*” represents the statistical significant difference between all the treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. “#” represents the statistical difference between “glutamate + ASH-WEX” treated groups with their respective “glutamate” treatment groups. “*” and “#” = p<0.05.

Article Snippet: C6 (rat glioma) and IMR-32 (human neuroblastoma) cells were purchased from National Centre for Cell Science (Pune, India).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunostaining, Control