Structured Review

Millipore 6 ohda
Effect of vehicle (n=29) and dose response of <t>6-OHDA</t> at 5 (n=18), 10 (n=24) and 14 μg/μl (n=10) on LC neuronal number and FST. A) 6-OHDA dose-response effect on LC neuronal number. *Indicates significant difference from vehicle-treated animals. #Indicates significant difference to 6-OHDA 14 μg/μl treated animals. B) 6-OHDA dose-response effect on FST immobile time (sec). *Indicates significant difference from vehicle-treated animals. Correlation of FST immobilization time (sec) to percent of control of LC neuronal number in vehicle- (C) and 6-OHDA- (D) treated animals. E) TH-IR labeling of LC neurons in vehicle and 6-OHDA treated animals at 5, 10 and 14 μg/μl.
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1) Product Images from "Depressive-like behavior observed with a minimal loss of locus coeruleus (LC) neurons following administration of 6-hydroxydopamine is associated with electrophysiological changes and reversed with precursors of norepinephrine"

Article Title: Depressive-like behavior observed with a minimal loss of locus coeruleus (LC) neurons following administration of 6-hydroxydopamine is associated with electrophysiological changes and reversed with precursors of norepinephrine

Journal: Neuropharmacology

doi: 10.1016/j.neuropharm.2015.09.003

Effect of vehicle (n=29) and dose response of 6-OHDA at 5 (n=18), 10 (n=24) and 14 μg/μl (n=10) on LC neuronal number and FST. A) 6-OHDA dose-response effect on LC neuronal number. *Indicates significant difference from vehicle-treated animals. #Indicates significant difference to 6-OHDA 14 μg/μl treated animals. B) 6-OHDA dose-response effect on FST immobile time (sec). *Indicates significant difference from vehicle-treated animals. Correlation of FST immobilization time (sec) to percent of control of LC neuronal number in vehicle- (C) and 6-OHDA- (D) treated animals. E) TH-IR labeling of LC neurons in vehicle and 6-OHDA treated animals at 5, 10 and 14 μg/μl.
Figure Legend Snippet: Effect of vehicle (n=29) and dose response of 6-OHDA at 5 (n=18), 10 (n=24) and 14 μg/μl (n=10) on LC neuronal number and FST. A) 6-OHDA dose-response effect on LC neuronal number. *Indicates significant difference from vehicle-treated animals. #Indicates significant difference to 6-OHDA 14 μg/μl treated animals. B) 6-OHDA dose-response effect on FST immobile time (sec). *Indicates significant difference from vehicle-treated animals. Correlation of FST immobilization time (sec) to percent of control of LC neuronal number in vehicle- (C) and 6-OHDA- (D) treated animals. E) TH-IR labeling of LC neurons in vehicle and 6-OHDA treated animals at 5, 10 and 14 μg/μl.

Techniques Used: Size-exclusion Chromatography, Labeling

Sucrose consumption in vehicle- (n=9) and 6-OHDA-treated (n=8) (5 μg/μl) animals. (A) Sucrose consumed (g) at the pre-test time and 1, 2 and 3 weeks after administration of vehicle or 6-OHDA. * Indicates significant difference from vehicle-treated animals at the pre-test point. # Indicates significant difference to vehicle- treated animals at the 3-week time point. (B) FST performed on the animals 4 days after the 3-week sucrose consumption test and (C) number of LC noradrenergic neurons in vehicle- and 6-OHDA-treated animals by TH-IH. * Indicates significant difference from vehicle-treated animals.
Figure Legend Snippet: Sucrose consumption in vehicle- (n=9) and 6-OHDA-treated (n=8) (5 μg/μl) animals. (A) Sucrose consumed (g) at the pre-test time and 1, 2 and 3 weeks after administration of vehicle or 6-OHDA. * Indicates significant difference from vehicle-treated animals at the pre-test point. # Indicates significant difference to vehicle- treated animals at the 3-week time point. (B) FST performed on the animals 4 days after the 3-week sucrose consumption test and (C) number of LC noradrenergic neurons in vehicle- and 6-OHDA-treated animals by TH-IH. * Indicates significant difference from vehicle-treated animals.

Techniques Used:

LC neuronal loss induced by 6-OHDA (5 μg/μl) altered electrophysiological properties of LC neurons. (A) Firing frequency, (B) coefficient of variation, (C) percent of LC neurons demonstrating burst activity, (D) mean number of spikes/burst activity, (E) percent of the number of spikes in a burst activity, (F) the number of spontaneously active LC neurons identified per tract and (G) LC neuronal number in vehicle- and 6-OHDA-treated animals by TH-IH. Seventy nine neurons were recorded in 4 vehicle-treated mice and 67 neurons in 6 mice bilaterally lesioned with 6-OHDA. * Indicates significant difference from vehicle-treated animals.
Figure Legend Snippet: LC neuronal loss induced by 6-OHDA (5 μg/μl) altered electrophysiological properties of LC neurons. (A) Firing frequency, (B) coefficient of variation, (C) percent of LC neurons demonstrating burst activity, (D) mean number of spikes/burst activity, (E) percent of the number of spikes in a burst activity, (F) the number of spontaneously active LC neurons identified per tract and (G) LC neuronal number in vehicle- and 6-OHDA-treated animals by TH-IH. Seventy nine neurons were recorded in 4 vehicle-treated mice and 67 neurons in 6 mice bilaterally lesioned with 6-OHDA. * Indicates significant difference from vehicle-treated animals.

Techniques Used: Activity Assay, Mouse Assay

Timeline of LC injection of vehicle or 6-OHDA to (A) FST and electrophysiological studies, (B) sucrose consumption test, and (C) administration of L-DOPA and DOPS.
Figure Legend Snippet: Timeline of LC injection of vehicle or 6-OHDA to (A) FST and electrophysiological studies, (B) sucrose consumption test, and (C) administration of L-DOPA and DOPS.

Techniques Used: Injection

2) Product Images from "Necdin and TrkA Contribute to Modulation by p75NTR of Resistance to Oxidant Stress"

Article Title: Necdin and TrkA Contribute to Modulation by p75NTR of Resistance to Oxidant Stress

Journal: Experimental cell research

doi: 10.1016/j.yexcr.2009.10.001

The effect of necdin siRNA on cell membrane integrity following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Trypan blue exclusion determinations were performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to those obtained in the absence of 6-OHDA for all conditions. Both p75NTR-native and p75NTR-deficient cell lines were treated with necdin or a scrambled control siRNA along with 6-OHDA treatment. *P
Figure Legend Snippet: The effect of necdin siRNA on cell membrane integrity following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Trypan blue exclusion determinations were performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to those obtained in the absence of 6-OHDA for all conditions. Both p75NTR-native and p75NTR-deficient cell lines were treated with necdin or a scrambled control siRNA along with 6-OHDA treatment. *P

Techniques Used: Concentration Assay

The effect of necdin siRNA and a JNK phosphorylation inhibitor, SP600125, on cell metabolic viability following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Alamar blue staining was performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. p75NTR-native and p75NTR-deficient cell lines were treated with necdin siRNA or a scrambled control siRNA with or without SP600125 and 6-OHDA treatment. *P values
Figure Legend Snippet: The effect of necdin siRNA and a JNK phosphorylation inhibitor, SP600125, on cell metabolic viability following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Alamar blue staining was performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. p75NTR-native and p75NTR-deficient cell lines were treated with necdin siRNA or a scrambled control siRNA with or without SP600125 and 6-OHDA treatment. *P values

Techniques Used: Staining, Concentration Assay

The effect of necdin siRNA on metabolic viability following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Alamar blue staining was performed as described in Materials and Methods and OD 590 nm values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. Both p75NTR-native and p75NTR-deficient cell lines were treated with necdin siRNA or a scrambled control siRNA along with 6-OHDA. *P
Figure Legend Snippet: The effect of necdin siRNA on metabolic viability following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Alamar blue staining was performed as described in Materials and Methods and OD 590 nm values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. Both p75NTR-native and p75NTR-deficient cell lines were treated with necdin siRNA or a scrambled control siRNA along with 6-OHDA. *P

Techniques Used: Staining, Concentration Assay

The effect of necdin and p75NTR siRNAs on metabolic viability following 6-OHDA treatment of p75NTR-native PC12 cells Alamar blue staining was performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. p75NTR-native cells were treated with necdin siRNA, p75NTR siRNA, or a scrambled control siRNA along with 6-OHDA treatment. *P
Figure Legend Snippet: The effect of necdin and p75NTR siRNAs on metabolic viability following 6-OHDA treatment of p75NTR-native PC12 cells Alamar blue staining was performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. p75NTR-native cells were treated with necdin siRNA, p75NTR siRNA, or a scrambled control siRNA along with 6-OHDA treatment. *P

Techniques Used: Staining, Concentration Assay

3) Product Images from "Decomposition of abnormal free locomotor behavior in a rat model of Parkinson's disease"

Article Title: Decomposition of abnormal free locomotor behavior in a rat model of Parkinson's disease

Journal: Frontiers in Systems Neuroscience

doi: 10.3389/fnsys.2013.00095

Circadian post-lesion activity . Free locomotor activity of all vehicle (A) and all 6-OHDA (B) injected rats after lesioning. Animal numbers are given left to the respective vertical bars representing global activity phases. Red solid lines mark light switches at 11 pm (lights on) and 11 am (lights off) that would take place under usual housing conditions. Note that behavioral monitoring was performed under constant dark conditions.
Figure Legend Snippet: Circadian post-lesion activity . Free locomotor activity of all vehicle (A) and all 6-OHDA (B) injected rats after lesioning. Animal numbers are given left to the respective vertical bars representing global activity phases. Red solid lines mark light switches at 11 pm (lights on) and 11 am (lights off) that would take place under usual housing conditions. Note that behavioral monitoring was performed under constant dark conditions.

Techniques Used: Activity Assay, Injection

Distribution of maximal speed values . Density functions for logarithmic values of maximal speeds (log max-SD) are shown for all control (left column) and all 6-OHDA lesioned rats (right column). Solid and dashed lines denote log max-SD distributions before and after lesioning, respectively. Animal numbers are given in the right top corner. log max-SD, base 10 logarithm of maximal speed in given movement episode; 6-OHDA, 6-hydroxydopamine.
Figure Legend Snippet: Distribution of maximal speed values . Density functions for logarithmic values of maximal speeds (log max-SD) are shown for all control (left column) and all 6-OHDA lesioned rats (right column). Solid and dashed lines denote log max-SD distributions before and after lesioning, respectively. Animal numbers are given in the right top corner. log max-SD, base 10 logarithm of maximal speed in given movement episode; 6-OHDA, 6-hydroxydopamine.

Techniques Used:

Bootstrap regressions. (A) Independent bootstrap regressions ( n = 55) of the 6-OHDA group ( n = 8 animals). The bootstrapping R -value is color-coded and clipped for correlations with P > 0.1 after FDR-correction (green fields). (B1–5) Correlations with P
Figure Legend Snippet: Bootstrap regressions. (A) Independent bootstrap regressions ( n = 55) of the 6-OHDA group ( n = 8 animals). The bootstrapping R -value is color-coded and clipped for correlations with P > 0.1 after FDR-correction (green fields). (B1–5) Correlations with P

Techniques Used:

Stereological counting of TH-positive SNc and VTA neurons. (A) Representative photomicrographs of TH-stained midbrain sections of a vehicle and 6-OHDA injected rat (left column, scale bar equals 500 μm) as well as 40× magnifications of VTA and SNc-ROIs (middle and right column, scale bar equals 50 μm). (B) 3D-reconstructions of stereological regions of interest for the same vehicle and 6-OHDA injected rats as in (A) . Red spheres indicate stereologically counted cells in the SNc and VTA. (C) Estimated absolute population of cells in the SNc and VTA for vehicle ( n = 5) and 6-OHDA ( n = 8) injected rats. Double asterisks denote P ≤ 0.001 (uncorrected). TH, tyrosine-hydroxylase; 6-OHDA, 6-hydroxydopamine; VTA, ventral tegmental area; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata.
Figure Legend Snippet: Stereological counting of TH-positive SNc and VTA neurons. (A) Representative photomicrographs of TH-stained midbrain sections of a vehicle and 6-OHDA injected rat (left column, scale bar equals 500 μm) as well as 40× magnifications of VTA and SNc-ROIs (middle and right column, scale bar equals 50 μm). (B) 3D-reconstructions of stereological regions of interest for the same vehicle and 6-OHDA injected rats as in (A) . Red spheres indicate stereologically counted cells in the SNc and VTA. (C) Estimated absolute population of cells in the SNc and VTA for vehicle ( n = 5) and 6-OHDA ( n = 8) injected rats. Double asterisks denote P ≤ 0.001 (uncorrected). TH, tyrosine-hydroxylase; 6-OHDA, 6-hydroxydopamine; VTA, ventral tegmental area; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata.

Techniques Used: Staining, Injection

4) Product Images from "The Parkinsonian mimetic, 6-OHDA, impairs axonal transport in dopaminergic axons"

Article Title: The Parkinsonian mimetic, 6-OHDA, impairs axonal transport in dopaminergic axons

Journal: Molecular Neurodegeneration

doi: 10.1186/1750-1326-9-17

6-OHDA induces microtubule disruption and retrograde degeneration in vitro. A) Integrity of microtubule tracks was assessed by measuring tubulin fragmentation in DA cultures treated with 6-OHDA at the indicated times and then fixed and stained with antibodies against AcTub and TH. Significant fragmentation of AcTub is not seen prior to 6 hours, but is readily apparent at 24 hours. B) TH positive axons with fragmented AcTub staining were quantified. One hundred to three hundred DA-GFP axons were counted per dish and 4–5 dishes were used per group. Scale bars indicate 10 μm. Bars represent mean ± SEM. ** indicates p
Figure Legend Snippet: 6-OHDA induces microtubule disruption and retrograde degeneration in vitro. A) Integrity of microtubule tracks was assessed by measuring tubulin fragmentation in DA cultures treated with 6-OHDA at the indicated times and then fixed and stained with antibodies against AcTub and TH. Significant fragmentation of AcTub is not seen prior to 6 hours, but is readily apparent at 24 hours. B) TH positive axons with fragmented AcTub staining were quantified. One hundred to three hundred DA-GFP axons were counted per dish and 4–5 dishes were used per group. Scale bars indicate 10 μm. Bars represent mean ± SEM. ** indicates p

Techniques Used: In Vitro, Staining

6-OHDA rapidly decreases mitochondrial movement in non-DA axons. A) Axonal movement of mitochondria in control and 6-OHDA treated axons. Non-GFP positive axons (non-DA; Top panels) that were labeled with MitoDsRed2 (Middle panels) were selected for imaging 30 minutes after treatment with 6-OHDA. Resulting kymographs are shown below. For additional clarity tracks of moving particles are depicted in the bottom panels: blue lines denote anterograde movement and red lines indicate retrograde trafficking. Scale bar indicates 10 μm. Quantification of B) moving mitochondria in both anterograde and retrograde directions (n = 3–4 devices per group from with 3–5 axons analyzed per device) and C) mitochondrial speeds of motile mitochondria. The latter were calculated as described [ 10 ] (n = 90–120 mitochondria per group). In B and C, data are represented as mean ± SEM, *: indicate p
Figure Legend Snippet: 6-OHDA rapidly decreases mitochondrial movement in non-DA axons. A) Axonal movement of mitochondria in control and 6-OHDA treated axons. Non-GFP positive axons (non-DA; Top panels) that were labeled with MitoDsRed2 (Middle panels) were selected for imaging 30 minutes after treatment with 6-OHDA. Resulting kymographs are shown below. For additional clarity tracks of moving particles are depicted in the bottom panels: blue lines denote anterograde movement and red lines indicate retrograde trafficking. Scale bar indicates 10 μm. Quantification of B) moving mitochondria in both anterograde and retrograde directions (n = 3–4 devices per group from with 3–5 axons analyzed per device) and C) mitochondrial speeds of motile mitochondria. The latter were calculated as described [ 10 ] (n = 90–120 mitochondria per group). In B and C, data are represented as mean ± SEM, *: indicate p

Techniques Used: Labeling, Imaging

6-OHDA rapidly depolarizes mitochondria in both DA and non-DA axons. A) To ensure rapid, even labeling of mitochondria with TMRE (25 nM), axons were assessed after they had exited the microdevice channels. Scale bar indicates 10 μm. B) 6-OHDA significantly decreased mitochondrial membrane potential (ΔΨm) in DA and non-DA axons. Data indicate mean ± SEM from four independent experiments (n = 18–30 axons per group). ** indicates p
Figure Legend Snippet: 6-OHDA rapidly depolarizes mitochondria in both DA and non-DA axons. A) To ensure rapid, even labeling of mitochondria with TMRE (25 nM), axons were assessed after they had exited the microdevice channels. Scale bar indicates 10 μm. B) 6-OHDA significantly decreased mitochondrial membrane potential (ΔΨm) in DA and non-DA axons. Data indicate mean ± SEM from four independent experiments (n = 18–30 axons per group). ** indicates p

Techniques Used: Labeling

6-OHDA also decreases synaptic vesicle movement in DA axons. A) DA-GFP cultures (Top panels) in microdevices were transduced with Syn-Cer lentivirus (Middle panels) at DIV2. Vesicular movement was assessed on DIV12–13 before and after toxin treatment. Resulting kymographs are shown below. Because of the smaller size of vesicular particles and the relative “dimness” of the cerulean emission, tracks of moving particles are shown in bottom panels for clarity. Red represents retrograde movement whereas blue is anterograde trafficking. B) Quantification of moving vesicles was determined as described in Materials and Methods. Scale bar: 5 μm. Mean ± SEM, total of 8 (control) and 8 (6-OHDA-treated) axons from 5–7 devices per group. * indicates p
Figure Legend Snippet: 6-OHDA also decreases synaptic vesicle movement in DA axons. A) DA-GFP cultures (Top panels) in microdevices were transduced with Syn-Cer lentivirus (Middle panels) at DIV2. Vesicular movement was assessed on DIV12–13 before and after toxin treatment. Resulting kymographs are shown below. Because of the smaller size of vesicular particles and the relative “dimness” of the cerulean emission, tracks of moving particles are shown in bottom panels for clarity. Red represents retrograde movement whereas blue is anterograde trafficking. B) Quantification of moving vesicles was determined as described in Materials and Methods. Scale bar: 5 μm. Mean ± SEM, total of 8 (control) and 8 (6-OHDA-treated) axons from 5–7 devices per group. * indicates p

Techniques Used: Transduction

Autophagy precedes cell death in midbrain neurons following 6-OHDA treatment. A) Autophagy was assessed by introducing a GFP-tagged LC3 expression clone at DIV6 and treating midbrain cultures 1 d later with 6-OHDA. LC3-positive puncta (arrows) were assessed by GFP fluorescence in representative neurons in control and after toxin treatment. B) The number of cells with at least three LC3-GFP puncta were counted and expressed as percentage of all neurons that were LC3-GFP positive, regardless of whether the LC3-GFP signal in these neurons was diffuse or punctated. Scale bar indicates 10 μm. Mean ± SEM from 3 independent experiments (n = 3–5 per group), * p
Figure Legend Snippet: Autophagy precedes cell death in midbrain neurons following 6-OHDA treatment. A) Autophagy was assessed by introducing a GFP-tagged LC3 expression clone at DIV6 and treating midbrain cultures 1 d later with 6-OHDA. LC3-positive puncta (arrows) were assessed by GFP fluorescence in representative neurons in control and after toxin treatment. B) The number of cells with at least three LC3-GFP puncta were counted and expressed as percentage of all neurons that were LC3-GFP positive, regardless of whether the LC3-GFP signal in these neurons was diffuse or punctated. Scale bar indicates 10 μm. Mean ± SEM from 3 independent experiments (n = 3–5 per group), * p

Techniques Used: Expressing, Fluorescence

6-OHDA rapidly decreases mitochondrial movement in DA axons. A) Diagram of microdevice B) Axonal movement of mitochondria in control and 6-OHDA treated axons. DA-GFP cultures (Top panels) grown in microdevices and transduced with MitoDsRed2 (Middle panels) were imaged 30 minutes after treatment with 6-OHDA. Resulting kymographs are shown below. For additional clarity tracks of moving particles are depicted in the bottom panels: blue lines denote anterograde movement and red lines indicate retrograde trafficking. Scale bar indicates 10 μm. Quantification of C) moving mitochondria (n = 4–5 devices per group with 4–5 axons analyzed per device) and D) mitochondrial speeds. The latter were calculated as described [ 10 ] (n = 60–80 mitochondria per group). In C and D , data are represented as mean ± SEM, * + indicates p
Figure Legend Snippet: 6-OHDA rapidly decreases mitochondrial movement in DA axons. A) Diagram of microdevice B) Axonal movement of mitochondria in control and 6-OHDA treated axons. DA-GFP cultures (Top panels) grown in microdevices and transduced with MitoDsRed2 (Middle panels) were imaged 30 minutes after treatment with 6-OHDA. Resulting kymographs are shown below. For additional clarity tracks of moving particles are depicted in the bottom panels: blue lines denote anterograde movement and red lines indicate retrograde trafficking. Scale bar indicates 10 μm. Quantification of C) moving mitochondria (n = 4–5 devices per group with 4–5 axons analyzed per device) and D) mitochondrial speeds. The latter were calculated as described [ 10 ] (n = 60–80 mitochondria per group). In C and D , data are represented as mean ± SEM, * + indicates p

Techniques Used: Transduction

5) Product Images from "Alterations of Sleep and Sleep Oscillations in the Hemiparkinsonian Rat"

Article Title: Alterations of Sleep and Sleep Oscillations in the Hemiparkinsonian Rat

Journal: Frontiers in Neuroscience

doi: 10.3389/fnins.2019.00148

Unilateral dopaminergic neuronal loss in the SNpc augmented theta amplitude across Wake, NREM and REM sleep in the MCx and hippocampus (Hipp) 14 (A) and 42 (B) days following the SNpc lesion caused by a microinfusion of 12 μg/1 μl 6-OHDA (12 μg/1 μl SNpc lesion) and 12 μg/2 μl 6-OHDA (12 μg/2 μl SNpc lesion). Theta amplitude augmentation following the unilateral SNpc lesion was particularly long-lasting in the MCx and Hipp during both Wake and REM sleep.
Figure Legend Snippet: Unilateral dopaminergic neuronal loss in the SNpc augmented theta amplitude across Wake, NREM and REM sleep in the MCx and hippocampus (Hipp) 14 (A) and 42 (B) days following the SNpc lesion caused by a microinfusion of 12 μg/1 μl 6-OHDA (12 μg/1 μl SNpc lesion) and 12 μg/2 μl 6-OHDA (12 μg/2 μl SNpc lesion). Theta amplitude augmentation following the unilateral SNpc lesion was particularly long-lasting in the MCx and Hipp during both Wake and REM sleep.

Techniques Used:

Wake episode dynamics in the MCx and hippocampus (Hipp) at 14 and at 42 days following the SNpc lesion caused by a microinfusion of 12 μg/1 μl 6-OHDA and 12 μg/2 μl 6-OHDA in contrast to the implanted controls (Control-i) - the group distributions of the mean number/6 h of Wake episodes over their durations (minutes). In these log-log distributions each “horizontal stair” is placed above its corresponding duration, depicted on the x -axis in minutes – e.g., the group mean number/6 h of 10 s episodes is placed above 10/60 = 0.17 min and so on. The first three episode durations are indicated below the “stair lines” of the two upper panels as 10, 20, 30, presenting the group mean number/6 h of the Wake 10, 20, and 30 s episodes. The missing lines in the distributions depict the zero group mean number of episodes that causes the logarithms to be identified as “not a number.”
Figure Legend Snippet: Wake episode dynamics in the MCx and hippocampus (Hipp) at 14 and at 42 days following the SNpc lesion caused by a microinfusion of 12 μg/1 μl 6-OHDA and 12 μg/2 μl 6-OHDA in contrast to the implanted controls (Control-i) - the group distributions of the mean number/6 h of Wake episodes over their durations (minutes). In these log-log distributions each “horizontal stair” is placed above its corresponding duration, depicted on the x -axis in minutes – e.g., the group mean number/6 h of 10 s episodes is placed above 10/60 = 0.17 min and so on. The first three episode durations are indicated below the “stair lines” of the two upper panels as 10, 20, 30, presenting the group mean number/6 h of the Wake 10, 20, and 30 s episodes. The missing lines in the distributions depict the zero group mean number of episodes that causes the logarithms to be identified as “not a number.”

Techniques Used:

Functional coupling between the dopaminergic neuronal loss of the substantia nigra pars compacta (SNpc) and theta amplitude during Wake, NREM and REM in the MCx. The s ignificant positive linear correlations ( r ≥ 0.51; p ≤ 0.05) between the Wake/NREM/REM relative theta amplitudes in the MCx and the dopaminergic (DA) neuronal loss throughout the overall SNpc rostro-caudal dimension at 14 (A) and 42 (B) days following the SNpc lesion caused by 12 μg/1 μl 6-OHDA (12 μg/1 μl SNpc lesion).
Figure Legend Snippet: Functional coupling between the dopaminergic neuronal loss of the substantia nigra pars compacta (SNpc) and theta amplitude during Wake, NREM and REM in the MCx. The s ignificant positive linear correlations ( r ≥ 0.51; p ≤ 0.05) between the Wake/NREM/REM relative theta amplitudes in the MCx and the dopaminergic (DA) neuronal loss throughout the overall SNpc rostro-caudal dimension at 14 (A) and 42 (B) days following the SNpc lesion caused by 12 μg/1 μl 6-OHDA (12 μg/1 μl SNpc lesion).

Techniques Used: Functional Assay

Coherence spectra of all the conventional EEG frequency oscillations between the MCx and hippocampus (Hipp) during Wake, NREM and REM sleep 14 and 42 days following the unilateral SNpc lesions with two distinct microinfusions of 6-OHDA. Arrows indicate the statistically significant mean coherence values at p ≤ 0.05, obtained using the Mann–Whitney U tests, while their directions indicate the increased or decreased coherences following the unilateral SNpc lesions vs. controls.
Figure Legend Snippet: Coherence spectra of all the conventional EEG frequency oscillations between the MCx and hippocampus (Hipp) during Wake, NREM and REM sleep 14 and 42 days following the unilateral SNpc lesions with two distinct microinfusions of 6-OHDA. Arrows indicate the statistically significant mean coherence values at p ≤ 0.05, obtained using the Mann–Whitney U tests, while their directions indicate the increased or decreased coherences following the unilateral SNpc lesions vs. controls.

Techniques Used: MANN-WHITNEY

Tyrosine hydroxilase (TH) immunohistochemical identification and quantification of the unilateral substantia nigra pars compacta (SNpc) dopaminergic neuronal loss. Quantified dopaminergic neuronal loss in the unilateral SNpc lesion caused by the microinfusion of 12 μg/1 μl 6-OHDA (A) and 12 μg/2 μl 6-OHDA (B) . Individual example of three sections used for the quantification of each SNpc lesioned brain (C–E) , representing three defined stereotaxic ranges with an identified SNpc partial lesion, caused by 12 μg/1 μl 6-OHDA, throughout the overall rostro-caudal SNpc dimension. The dopaminergic neuronal loss in this SNpc was: 62.30% from 4.6 to 5.1 mm caudally from bregma; 74.47% from 5.2 to 5.7 mm caudally from bregma; and 93.34% from 5.8 to 6.3 mm caudally from bregma. VTA, ventral tegmental area; ∗ – indicates the lesioned SNpc; Scale bar 200 μm. The scale bars of the upper (C–E) panels, representing only the typical brain sections for the specific SNpc stereotaxic range, are not defined, since they were obtained by the acquisition of the images by using the 10× objective lens of the Olympus BX-51 microscope, equipped with a motorized stage and CCD video camera (Pixelink, Ottawa, ON, Canada), and by using a superimage acquisition option within the newCAST stereological software package (VIS-Visiopharm Integrator System, version 5.3.1.1640; Visiopharm; Denmark).
Figure Legend Snippet: Tyrosine hydroxilase (TH) immunohistochemical identification and quantification of the unilateral substantia nigra pars compacta (SNpc) dopaminergic neuronal loss. Quantified dopaminergic neuronal loss in the unilateral SNpc lesion caused by the microinfusion of 12 μg/1 μl 6-OHDA (A) and 12 μg/2 μl 6-OHDA (B) . Individual example of three sections used for the quantification of each SNpc lesioned brain (C–E) , representing three defined stereotaxic ranges with an identified SNpc partial lesion, caused by 12 μg/1 μl 6-OHDA, throughout the overall rostro-caudal SNpc dimension. The dopaminergic neuronal loss in this SNpc was: 62.30% from 4.6 to 5.1 mm caudally from bregma; 74.47% from 5.2 to 5.7 mm caudally from bregma; and 93.34% from 5.8 to 6.3 mm caudally from bregma. VTA, ventral tegmental area; ∗ – indicates the lesioned SNpc; Scale bar 200 μm. The scale bars of the upper (C–E) panels, representing only the typical brain sections for the specific SNpc stereotaxic range, are not defined, since they were obtained by the acquisition of the images by using the 10× objective lens of the Olympus BX-51 microscope, equipped with a motorized stage and CCD video camera (Pixelink, Ottawa, ON, Canada), and by using a superimage acquisition option within the newCAST stereological software package (VIS-Visiopharm Integrator System, version 5.3.1.1640; Visiopharm; Denmark).

Techniques Used: Immunohistochemistry, Microscopy, Software

6) Product Images from "The sympathetic nervous system modulates CD4+FoxP3+ regulatory T cells via a TGF-?-dependent mechanism"

Article Title: The sympathetic nervous system modulates CD4+FoxP3+ regulatory T cells via a TGF-?-dependent mechanism

Journal: Journal of Leukocyte Biology

doi: 10.1189/jlb.0209107

Abrogation of DTH reaction in 6-OHDA-treated, immunized mice. Mice were immunized with MOG/CFA 2 days after receiving i.p. dose of 6-OHDA (200 mg/kg). Control groups did not receive any immunization. Seven days after immunizing, one footpad was challenged with the MOG and the other footpad with vehicle only. (A) The thickness of footpads measured 24 h after challenge. Bar graph represents the mean ± SE footpad swelling of 6 mice/group, 2 experiments. (B) Histology of the footpad 24 h after challenge with MOG from immunized mice without challenge (Control), immunized mice that received an i.p. injection of PBS or 6-OHDA 2 days before immunization and then challenged. Mice showed perivascular infiltration of cells in the PBS or 6-OHDA-treated groups. Magnification ×20.
Figure Legend Snippet: Abrogation of DTH reaction in 6-OHDA-treated, immunized mice. Mice were immunized with MOG/CFA 2 days after receiving i.p. dose of 6-OHDA (200 mg/kg). Control groups did not receive any immunization. Seven days after immunizing, one footpad was challenged with the MOG and the other footpad with vehicle only. (A) The thickness of footpads measured 24 h after challenge. Bar graph represents the mean ± SE footpad swelling of 6 mice/group, 2 experiments. (B) Histology of the footpad 24 h after challenge with MOG from immunized mice without challenge (Control), immunized mice that received an i.p. injection of PBS or 6-OHDA 2 days before immunization and then challenged. Mice showed perivascular infiltration of cells in the PBS or 6-OHDA-treated groups. Magnification ×20.

Techniques Used: Mouse Assay, Injection

6-OHDA induced sympathectomy does not change the frequency of total CD4+ T cells . C57BL/6 mice were treated with 6-OHDA, while control mice received PBS. Forty-eight hours after injection, total spleen cells were stained. Representative FACS dot plots demonstrate the percentage of CD4 + CD25 + T cells in the spleen, 48 h after treatment. Plots represent a total lymphocyte gate. The values represent different cell types, as a percentage of total lymphocytes.
Figure Legend Snippet: 6-OHDA induced sympathectomy does not change the frequency of total CD4+ T cells . C57BL/6 mice were treated with 6-OHDA, while control mice received PBS. Forty-eight hours after injection, total spleen cells were stained. Representative FACS dot plots demonstrate the percentage of CD4 + CD25 + T cells in the spleen, 48 h after treatment. Plots represent a total lymphocyte gate. The values represent different cell types, as a percentage of total lymphocytes.

Techniques Used: Mouse Assay, Injection, Staining, FACS

Peripheral chemical sympathectomy by 6-OHDA induces an increase in the number of Tregs. (A) Representative dot plots show flow cytometric analysis of mouse (C57BL/6) spleen and inguinal lymph node cells at 48-h post injection of PBS, 6-OHDA, or desipramine and 6-OHDA. Plots represent gated CD4 T cells. The values in the upper-right quadrant represent CD4 + FoxP3 + T cells as a percentage of total CD4 T cells. (B) Bar graph shows the mean results ( n =12–15). (C) Bar graph showing the absolute number of CD4 + FoxP3 + Tregs from the above experiment. Numbers were calculated from flow cytometry data from the spleen. (D) Propranolol fails to prevent the increase in Tregs by chemical sympathectomy: representative dot plots show flow cytometric analysis of mouse (C57BL/6) spleen and inguinal lymph node cells at 48 h postinjection of PBS, 6-OHDA, or 6-OHDA followed by propranolol. Plots represent gated CD4 T cells. The values in the upper-right quadrant represent CD4 + FoxP3 + T cells as a percentage of total CD4 T cells. (E) 6-OHDA does not effect the number of total CD8+ cells or CD8+ CD122+ cells. Representative dot plots show flow cytometric analysis of mouse (C57BL/6) spleen cells at 48 h postinjection of PBS or 6-OHDA. Cells are gated for CD3+. The values in the upper right quadrant represent CD8 + CD122 + T cells.
Figure Legend Snippet: Peripheral chemical sympathectomy by 6-OHDA induces an increase in the number of Tregs. (A) Representative dot plots show flow cytometric analysis of mouse (C57BL/6) spleen and inguinal lymph node cells at 48-h post injection of PBS, 6-OHDA, or desipramine and 6-OHDA. Plots represent gated CD4 T cells. The values in the upper-right quadrant represent CD4 + FoxP3 + T cells as a percentage of total CD4 T cells. (B) Bar graph shows the mean results ( n =12–15). (C) Bar graph showing the absolute number of CD4 + FoxP3 + Tregs from the above experiment. Numbers were calculated from flow cytometry data from the spleen. (D) Propranolol fails to prevent the increase in Tregs by chemical sympathectomy: representative dot plots show flow cytometric analysis of mouse (C57BL/6) spleen and inguinal lymph node cells at 48 h postinjection of PBS, 6-OHDA, or 6-OHDA followed by propranolol. Plots represent gated CD4 T cells. The values in the upper-right quadrant represent CD4 + FoxP3 + T cells as a percentage of total CD4 T cells. (E) 6-OHDA does not effect the number of total CD8+ cells or CD8+ CD122+ cells. Representative dot plots show flow cytometric analysis of mouse (C57BL/6) spleen cells at 48 h postinjection of PBS or 6-OHDA. Cells are gated for CD3+. The values in the upper right quadrant represent CD8 + CD122 + T cells.

Techniques Used: Flow Cytometry, Injection, Cytometry

6-OHDA-induced increase in Tregs is mediated by TGF-β. (A) TGF-β mRNA expression in PBS or 6-OHDA-treated C57BL/6 mice, assessed by qRT-PCR from total spleen cells taken 48 h after injection. Bar graph shows TGF-β mRNA expression as a percentage of RPL-19 (a housekeeping gene) expression. (B) Representative FACS dot plots demonstrating the percentage of CD4 + FoxP3 + Tregs in the spleens of PBS-treated or 6-OHDA treated TGFbRII transgenic and Cbl-b-(−/−) mouse, 48 h after treatment. Plots represent gated CD4 T cells. The values in the upper right quadrant represent CD4 + FoxP3 + T cells as a percentage of total CD4 T cells. (C) Bar graph shows the mean result ( n =10). (D) TGF-β mRNA expression before and after 6-OHDA induced peripheral sympathectomy in TGFbRII mice qRT-PCR from total spleen cells taken 48 h after injection. Bar graph shows TGF-β mRNA expression as percentage of RPL-19 expression.
Figure Legend Snippet: 6-OHDA-induced increase in Tregs is mediated by TGF-β. (A) TGF-β mRNA expression in PBS or 6-OHDA-treated C57BL/6 mice, assessed by qRT-PCR from total spleen cells taken 48 h after injection. Bar graph shows TGF-β mRNA expression as a percentage of RPL-19 (a housekeeping gene) expression. (B) Representative FACS dot plots demonstrating the percentage of CD4 + FoxP3 + Tregs in the spleens of PBS-treated or 6-OHDA treated TGFbRII transgenic and Cbl-b-(−/−) mouse, 48 h after treatment. Plots represent gated CD4 T cells. The values in the upper right quadrant represent CD4 + FoxP3 + T cells as a percentage of total CD4 T cells. (C) Bar graph shows the mean result ( n =10). (D) TGF-β mRNA expression before and after 6-OHDA induced peripheral sympathectomy in TGFbRII mice qRT-PCR from total spleen cells taken 48 h after injection. Bar graph shows TGF-β mRNA expression as percentage of RPL-19 expression.

Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Injection, FACS, Transgenic Assay

Tregs from 6-OHDA-treated mice are functionally similar to Tregs from PBS-treated mice. In vitro Treg functional assay: CD4 + CD25 – T cells (T effs) from the spleen of naïve C57BL/6 were labeled with CFSE, in each group. CFSE-labeled responder T effs did not proliferate in unstimulated conditions (No Stim.) but did proliferate in the presence of soluble anti-CD3 as demonstrated by the dilution of CFSE in the stimulated group. Tregs cocultured with T effs and antigen-presenting cells (live splenic DCs) in the presence of anti-CD3, as described, suppress the proliferation of responder T eff cells as quantified by CFSE dilution. Tregs were isolated from control or 6-OHDA-treated animals and cocultured with naïve T effs at a 1:1, 1:2, 1:4 Treg-to-T eff ratio.
Figure Legend Snippet: Tregs from 6-OHDA-treated mice are functionally similar to Tregs from PBS-treated mice. In vitro Treg functional assay: CD4 + CD25 – T cells (T effs) from the spleen of naïve C57BL/6 were labeled with CFSE, in each group. CFSE-labeled responder T effs did not proliferate in unstimulated conditions (No Stim.) but did proliferate in the presence of soluble anti-CD3 as demonstrated by the dilution of CFSE in the stimulated group. Tregs cocultured with T effs and antigen-presenting cells (live splenic DCs) in the presence of anti-CD3, as described, suppress the proliferation of responder T eff cells as quantified by CFSE dilution. Tregs were isolated from control or 6-OHDA-treated animals and cocultured with naïve T effs at a 1:1, 1:2, 1:4 Treg-to-T eff ratio.

Techniques Used: Mouse Assay, In Vitro, Functional Assay, Labeling, Isolation

6-OHDA-induced peripheral chemical sympathectomy prevents EAE . (A) Female C57BL/6 mice, 7 to 8 wk old were injected with 6-OHDA, and then, 48 h later were immunized with MOG, as described previously. Clinical scores of vehicle-treated mice (light shade) and mice treated with 6-OHDA (dark shade) (0, normal; 1, limp tail; 2, paraparesis with a clumsy gait; 3, hind limb paralysis; 4, quadriplegia; 5, death) were assessed each day. Data represent mean scores ( n =10, vehicle and; n =12, 6-OHDA) ± SE. (B) Mice were immunized with MOG peptide and received 1 × 10 6 CD4 + T cells (i.v.) from PBS or 6-OHDA-treated mice immediately after immunization. Clinical scores of uninjected (black), PBS-CD4 + T cells-injected (—▾—) and 6-OHDA- CD4 + T cells-injected (light shade) mice, assessed each day. Data represent mean scores ( n =10, uninjected; n =10, PBS-CD4 + T cells injected; and n =12, 6-OHDA-CD4 + T cells injected).
Figure Legend Snippet: 6-OHDA-induced peripheral chemical sympathectomy prevents EAE . (A) Female C57BL/6 mice, 7 to 8 wk old were injected with 6-OHDA, and then, 48 h later were immunized with MOG, as described previously. Clinical scores of vehicle-treated mice (light shade) and mice treated with 6-OHDA (dark shade) (0, normal; 1, limp tail; 2, paraparesis with a clumsy gait; 3, hind limb paralysis; 4, quadriplegia; 5, death) were assessed each day. Data represent mean scores ( n =10, vehicle and; n =12, 6-OHDA) ± SE. (B) Mice were immunized with MOG peptide and received 1 × 10 6 CD4 + T cells (i.v.) from PBS or 6-OHDA-treated mice immediately after immunization. Clinical scores of uninjected (black), PBS-CD4 + T cells-injected (—▾—) and 6-OHDA- CD4 + T cells-injected (light shade) mice, assessed each day. Data represent mean scores ( n =10, uninjected; n =10, PBS-CD4 + T cells injected; and n =12, 6-OHDA-CD4 + T cells injected).

Techniques Used: Mouse Assay, Injection

7) Product Images from "PI3 Kinase/Akt Activation Mediates Estrogen and IGF-1 Nigral DA Neuronal Neuroprotection Against a Unilateral Rat Model of Parkinson's Disease"

Article Title: PI3 Kinase/Akt Activation Mediates Estrogen and IGF-1 Nigral DA Neuronal Neuroprotection Against a Unilateral Rat Model of Parkinson's Disease

Journal:

doi: 10.1002/dneu.20609

Photomicrographs of TH immunoreactivity in the SNpc. PI3K and MAPK inhibitors were used to study estrogen and IGF-1 protection of DA neurons 7 days following unilateral injection of 6-hyroxydopamine (6-OHDA, 2.0 μ L of 4 μ g/ μ L)
Figure Legend Snippet: Photomicrographs of TH immunoreactivity in the SNpc. PI3K and MAPK inhibitors were used to study estrogen and IGF-1 protection of DA neurons 7 days following unilateral injection of 6-hyroxydopamine (6-OHDA, 2.0 μ L of 4 μ g/ μ L)

Techniques Used: Injection

Photomicrographs of striatal TH immunoreactivity 7 days after unilateral injection of 6-OHDA (2.0 μ L of 4 μ g/ μ L) into the medial forebrain bundle. A: Nonlesion control, (E) Lesion side. B: 17 β -estradiol benzoate (EB; 20
Figure Legend Snippet: Photomicrographs of striatal TH immunoreactivity 7 days after unilateral injection of 6-OHDA (2.0 μ L of 4 μ g/ μ L) into the medial forebrain bundle. A: Nonlesion control, (E) Lesion side. B: 17 β -estradiol benzoate (EB; 20

Techniques Used: Injection

A: Timeline of treatments; animals were given 17 β -estradiol benzoate (EB; 20 μ g, s.c) or vehicle 24 h before MFB 6-OHDA lesions. Lateral ventricle (icv) administration of IGF-1 (100 μ g/mL) or aCSF were administered 5 min after
Figure Legend Snippet: A: Timeline of treatments; animals were given 17 β -estradiol benzoate (EB; 20 μ g, s.c) or vehicle 24 h before MFB 6-OHDA lesions. Lateral ventricle (icv) administration of IGF-1 (100 μ g/mL) or aCSF were administered 5 min after

Techniques Used:

8) Product Images from "Parkinsonian Beta Oscillations in the External Globus Pallidus and Their Relationship with Subthalamic Nucleus Activity"

Article Title: Parkinsonian Beta Oscillations in the External Globus Pallidus and Their Relationship with Subthalamic Nucleus Activity

Journal:

doi: 10.1523/JNEUROSCI.4199-08.2008

Excessive synchronization of globus pallidus ensemble activity after 6-OHDA lesion is associated with decreases in the firing rate and regularity of single neurons.
Figure Legend Snippet: Excessive synchronization of globus pallidus ensemble activity after 6-OHDA lesion is associated with decreases in the firing rate and regularity of single neurons.

Techniques Used: Activity Assay

Spike timing of subthalamic nucleus and globus pallidus neurons in relation to cortical β oscillations in 6-OHDA-lesioned rats.
Figure Legend Snippet: Spike timing of subthalamic nucleus and globus pallidus neurons in relation to cortical β oscillations in 6-OHDA-lesioned rats.

Techniques Used:

Population analyses of globus pallidus neuron spike rate and timing in control and 6-OHDA-lesioned rats during cortical slow-wave activity.
Figure Legend Snippet: Population analyses of globus pallidus neuron spike rate and timing in control and 6-OHDA-lesioned rats during cortical slow-wave activity.

Techniques Used: Activity Assay

Brain state-dependency of the power and frequency of local field potentials in cortex and globus pallidus in control and 6-OHDA-lesioned rats.
Figure Legend Snippet: Brain state-dependency of the power and frequency of local field potentials in cortex and globus pallidus in control and 6-OHDA-lesioned rats.

Techniques Used:

Single-cell and network activity in the globus pallidus of control and 6-OHDA-lesioned rats during cortical activation.
Figure Legend Snippet: Single-cell and network activity in the globus pallidus of control and 6-OHDA-lesioned rats during cortical activation.

Techniques Used: Activity Assay, Activation Assay

Dichotomy of globus pallidus unit activity in 6-OHDA-lesioned rats is maintained across slow (~1 Hz) and β (~20 Hz) network oscillations.
Figure Legend Snippet: Dichotomy of globus pallidus unit activity in 6-OHDA-lesioned rats is maintained across slow (~1 Hz) and β (~20 Hz) network oscillations.

Techniques Used: Activity Assay

Single-cell and network activity in the globus pallidus of control and 6-OHDA-lesioned rats during cortical slow-wave activity.
Figure Legend Snippet: Single-cell and network activity in the globus pallidus of control and 6-OHDA-lesioned rats during cortical slow-wave activity.

Techniques Used: Activity Assay

9) Product Images from "Echinacoside's nigrostriatal dopaminergic protection against 6‐OHDA‐Induced endoplasmic reticulum stress through reducing the accumulation of Seipin"

Article Title: Echinacoside's nigrostriatal dopaminergic protection against 6‐OHDA‐Induced endoplasmic reticulum stress through reducing the accumulation of Seipin

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13285

ECH protected PC1 2 from 6‐OHDA‐induced ERS through promoting seipin ubiquitination and degradation. ( A ) ECH down‐regulated seipin and ERS‐associated protein expression induced by 6‐OHDA. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 3, 40 μg protein each lane). ( B ) Knocking down seipin by siRNA attenuated ERS‐associated protein accumulation induced by 6‐OHDA. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 3, 40 μg protein each lane). SiRNA, 50 nM each group transfected and collected to obtain protein lysis after 48 hrs. ( C ) The relative mRNA levels of seipin were determined using RT‐PCR. The results of RT‐PCR were normalized to GAPDH and expressed as fold change to control. SiRNA of seipin dramatically inhibited mRNA levels of seipin ( P
Figure Legend Snippet: ECH protected PC1 2 from 6‐OHDA‐induced ERS through promoting seipin ubiquitination and degradation. ( A ) ECH down‐regulated seipin and ERS‐associated protein expression induced by 6‐OHDA. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 3, 40 μg protein each lane). ( B ) Knocking down seipin by siRNA attenuated ERS‐associated protein accumulation induced by 6‐OHDA. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 3, 40 μg protein each lane). SiRNA, 50 nM each group transfected and collected to obtain protein lysis after 48 hrs. ( C ) The relative mRNA levels of seipin were determined using RT‐PCR. The results of RT‐PCR were normalized to GAPDH and expressed as fold change to control. SiRNA of seipin dramatically inhibited mRNA levels of seipin ( P

Techniques Used: Expressing, Western Blot, Transfection, Lysis, Reverse Transcription Polymerase Chain Reaction

ECH protected Nigrostriatal neurons from 6‐OHDA‐induced ERS‐associated proteins upregulation and inhibited seipin over accumulation in vivo . ( A ) Protein lysis solution of 5 rats’ striatum in each group pooled together. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 5, 40 μg protein each lane). ( B ) The immunoreactive bands were quantified by densitometry using ImageJ software and normalized to GAPDH. ( n = 3, * P
Figure Legend Snippet: ECH protected Nigrostriatal neurons from 6‐OHDA‐induced ERS‐associated proteins upregulation and inhibited seipin over accumulation in vivo . ( A ) Protein lysis solution of 5 rats’ striatum in each group pooled together. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 5, 40 μg protein each lane). ( B ) The immunoreactive bands were quantified by densitometry using ImageJ software and normalized to GAPDH. ( n = 3, * P

Techniques Used: In Vivo, Lysis, Expressing, Western Blot, Software

ECH protected Nigrostriatal neurons from 6‐OHDA‐induced TH downregulation and seipin overexpression in vivo and inhibited ERS‐associated protein Bip expression through seipin in vitro . ( A and B ) Immunofluorescence assay about brain SNpc slices in the model group and 7 mg/kg/day ECH group. Left, untreated side, Right, operation lesion side, Whole, the whole brain(2.5 × objective). The circle showed SNpc area of each side (20 × objective); ( A ) 6‐OHDA caused seipin accumulation and TH expression inhibited in SNpc. ( B ) ECH inhibited seipin and Bip overexpression induced by 6‐OHDA in SNpc. Blue, DAPI; green, Seipin; red, TH; scale bar, 50 μM. ( C and D ) Immunofluorescence assay about PC12 cells treated with different conditions. ( C ) ECH attenuated seipin and Bip expression in cytoplasm. ( D ) Modified seipin expression by siRNA could inhibit Bip overexpression induced by 6‐OHDA. Blue, DAPI; green, Seipin; red, Bip; scale bar, 50 μM.
Figure Legend Snippet: ECH protected Nigrostriatal neurons from 6‐OHDA‐induced TH downregulation and seipin overexpression in vivo and inhibited ERS‐associated protein Bip expression through seipin in vitro . ( A and B ) Immunofluorescence assay about brain SNpc slices in the model group and 7 mg/kg/day ECH group. Left, untreated side, Right, operation lesion side, Whole, the whole brain(2.5 × objective). The circle showed SNpc area of each side (20 × objective); ( A ) 6‐OHDA caused seipin accumulation and TH expression inhibited in SNpc. ( B ) ECH inhibited seipin and Bip overexpression induced by 6‐OHDA in SNpc. Blue, DAPI; green, Seipin; red, TH; scale bar, 50 μM. ( C and D ) Immunofluorescence assay about PC12 cells treated with different conditions. ( C ) ECH attenuated seipin and Bip expression in cytoplasm. ( D ) Modified seipin expression by siRNA could inhibit Bip overexpression induced by 6‐OHDA. Blue, DAPI; green, Seipin; red, Bip; scale bar, 50 μM.

Techniques Used: Over Expression, In Vivo, Expressing, In Vitro, Immunofluorescence, Modification

ECH rescued protein and mRNA expression levels of TH, DAT and decreased α‐synuclein accumulation in 6‐OHDA‐induced rat. ( A ) After intraperitoneally treated with vehicle or 0, 3.5, 7 mg/kg of ECH for 14 days, relative protein levels were measured using Western blotting ( n = 3), GAPDH taken as a reference gene. ( B ) The immunoreactive bands were quantified by densitometry using ImageJ software and normalized to GAPDH ( n = 3, * P
Figure Legend Snippet: ECH rescued protein and mRNA expression levels of TH, DAT and decreased α‐synuclein accumulation in 6‐OHDA‐induced rat. ( A ) After intraperitoneally treated with vehicle or 0, 3.5, 7 mg/kg of ECH for 14 days, relative protein levels were measured using Western blotting ( n = 3), GAPDH taken as a reference gene. ( B ) The immunoreactive bands were quantified by densitometry using ImageJ software and normalized to GAPDH ( n = 3, * P

Techniques Used: Expressing, Western Blot, Software

Ultrastructural evidence of ECH anti‐ERS (endoplasmic reticulum stress) effects as evaluated by transmission electron microscopy and ECH attenuated the 6‐OHDA‐induced intracellular ROS levels measured by flow cytometry. PC12 cells were pre‐incubated with different concentration of ECH (0, 25, 50, 100 μM) in a serum‐free RPMI‐1640 medium for 1 hr. Then, 6‐OHDA was added to the wells at a final concentration of 100 μM and incubated for another 24 hrs at 37°C. ( A ) 6‐OHDA‐induced ER curled and generated vacuole and ECH rescued ER structural integrity and made it extend. Black arrows, ER; scale bar, 0.5 μm. ( B ) ROS levels were determined using H2DCFH‐DA and measured by flow cytometry. ( C ) Data were calculated as ratio of fluorescence compared with untreated cells. Data are presented as mean ± S.D. values of three independent experiments. n = 3, * P
Figure Legend Snippet: Ultrastructural evidence of ECH anti‐ERS (endoplasmic reticulum stress) effects as evaluated by transmission electron microscopy and ECH attenuated the 6‐OHDA‐induced intracellular ROS levels measured by flow cytometry. PC12 cells were pre‐incubated with different concentration of ECH (0, 25, 50, 100 μM) in a serum‐free RPMI‐1640 medium for 1 hr. Then, 6‐OHDA was added to the wells at a final concentration of 100 μM and incubated for another 24 hrs at 37°C. ( A ) 6‐OHDA‐induced ER curled and generated vacuole and ECH rescued ER structural integrity and made it extend. Black arrows, ER; scale bar, 0.5 μm. ( B ) ROS levels were determined using H2DCFH‐DA and measured by flow cytometry. ( C ) Data were calculated as ratio of fluorescence compared with untreated cells. Data are presented as mean ± S.D. values of three independent experiments. n = 3, * P

Techniques Used: Transmission Assay, Electron Microscopy, Flow Cytometry, Cytometry, Incubation, Concentration Assay, Generated, Fluorescence

Protective effect of ECH on viability of PC12 cells injured by 6‐OHDA. Cell viability was determined by MTT assay. Data are expressed as percentage of the viability of cells, control taken as 100% viability. ( A ) The chemical structure of echinacoside (ECH). ( B ) Effect of different concentration of ECH on cell viability in PC12 cells (determine the non‐toxic dosages of ECH). PC12 cells were incubated with ECH (12.5–500 μM) for 24 hrs. ( C ) Effect of different concentration of 6‐OHDA on cell viability in PC12 cells (determine the toxic dosages of 6‐OHDA). PC12 cells were treated with various concentrations of 6‐OHDA (0–200 μM) for 24 hrs. ( D ) Effect of ECH on cell viability changes in 6‐OHDA‐induced PC12 cells. PC12 cells were pre‐incubated with different concentration of ECH (0, 25, 50, 100 μM) for 1 hr. Then, 6‐OHDA was added to the wells at a final concentration of 100 μM and incubated for another 24 hrs at 37°C. Data were shown as mean ± S.D., n = 4, * P
Figure Legend Snippet: Protective effect of ECH on viability of PC12 cells injured by 6‐OHDA. Cell viability was determined by MTT assay. Data are expressed as percentage of the viability of cells, control taken as 100% viability. ( A ) The chemical structure of echinacoside (ECH). ( B ) Effect of different concentration of ECH on cell viability in PC12 cells (determine the non‐toxic dosages of ECH). PC12 cells were incubated with ECH (12.5–500 μM) for 24 hrs. ( C ) Effect of different concentration of 6‐OHDA on cell viability in PC12 cells (determine the toxic dosages of 6‐OHDA). PC12 cells were treated with various concentrations of 6‐OHDA (0–200 μM) for 24 hrs. ( D ) Effect of ECH on cell viability changes in 6‐OHDA‐induced PC12 cells. PC12 cells were pre‐incubated with different concentration of ECH (0, 25, 50, 100 μM) for 1 hr. Then, 6‐OHDA was added to the wells at a final concentration of 100 μM and incubated for another 24 hrs at 37°C. Data were shown as mean ± S.D., n = 4, * P

Techniques Used: MTT Assay, Concentration Assay, Incubation

Nigrostriatal dopaminergic protection of ECH in 6‐OHDA‐induced rat. ( A ) ECH maintained neuronal morphology in striatum: vehicle group, black arrow, angioedema, caused by the surgery; 6‐OHDA group, green arrow, disordered fibre cord, black arrow, white matter oedema; ECH low concentration group, black arrow, white matter oedema; ECH‐high concentration group, no obvious oedema. ( B ) ECH rescued the number of tyrosine hydroxylase(TH)‐immunoreactive DA cells in the substantia nigra (SN) induced by 6‐OHDA damaged. Immunohistochemistry assay of TH taken in 4‐μm paraffin section of the SN. Brown region, TH‐immunoreactive DA cell bodies (20 × objective under white light). ( C ) Relative quantitative counting of TH‐immunoreactive DA cells in each group. The control group taken as 100 per cent compared with other groups ( n = 5, NS, No significant, * P
Figure Legend Snippet: Nigrostriatal dopaminergic protection of ECH in 6‐OHDA‐induced rat. ( A ) ECH maintained neuronal morphology in striatum: vehicle group, black arrow, angioedema, caused by the surgery; 6‐OHDA group, green arrow, disordered fibre cord, black arrow, white matter oedema; ECH low concentration group, black arrow, white matter oedema; ECH‐high concentration group, no obvious oedema. ( B ) ECH rescued the number of tyrosine hydroxylase(TH)‐immunoreactive DA cells in the substantia nigra (SN) induced by 6‐OHDA damaged. Immunohistochemistry assay of TH taken in 4‐μm paraffin section of the SN. Brown region, TH‐immunoreactive DA cell bodies (20 × objective under white light). ( C ) Relative quantitative counting of TH‐immunoreactive DA cells in each group. The control group taken as 100 per cent compared with other groups ( n = 5, NS, No significant, * P

Techniques Used: Concentration Assay, Immunohistochemistry, Paraffin Section

10) Product Images from "Electrophysiology and Pharmacology of Striatal Neuronal Dysfunction Induced by Mitochondrial Complex I Inhibition"

Article Title: Electrophysiology and Pharmacology of Striatal Neuronal Dysfunction Induced by Mitochondrial Complex I Inhibition

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1947-08.2008

The electrophysiological effects of rotenone are not affected in rats with complete striatal dopamine denervation by 6-OHDA and in mice transgenic for truncated human α-synuclein. A , Representative sections of TH (green) and NeuN (red) double immunostaining in rats with 6-OHDA-induced lesion of the substantia nigra pars compacta. On the left (ipsi) is shown the lesioned side, and on the right (contra) is shown the contralateral part. Scale bar, 100 μm. B , The graph shows that time course and the amplitude of the rotenone-induce electrophysiological effect were not different in DA-denervated and sham-operated rats. C , The effect of this toxin was similar in mice transgenic for truncated human α-synuclein (α-syn120 mice) and in wild-type mice.
Figure Legend Snippet: The electrophysiological effects of rotenone are not affected in rats with complete striatal dopamine denervation by 6-OHDA and in mice transgenic for truncated human α-synuclein. A , Representative sections of TH (green) and NeuN (red) double immunostaining in rats with 6-OHDA-induced lesion of the substantia nigra pars compacta. On the left (ipsi) is shown the lesioned side, and on the right (contra) is shown the contralateral part. Scale bar, 100 μm. B , The graph shows that time course and the amplitude of the rotenone-induce electrophysiological effect were not different in DA-denervated and sham-operated rats. C , The effect of this toxin was similar in mice transgenic for truncated human α-synuclein (α-syn120 mice) and in wild-type mice.

Techniques Used: Mouse Assay, Transgenic Assay, Double Immunostaining

11) Product Images from "The Neuroprotection of Low-Dose Morphine in Cellular and Animal Models of Parkinson’s Disease Through Ameliorating Endoplasmic Reticulum (ER) Stress and Activating Autophagy"

Article Title: The Neuroprotection of Low-Dose Morphine in Cellular and Animal Models of Parkinson’s Disease Through Ameliorating Endoplasmic Reticulum (ER) Stress and Activating Autophagy

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00120

The role of autophagy in morphine-induced neuroprotection in SH-SY5Y cells. (A) Double immunofluorescent staining revealed accumulation of LC3 punctuated foci in cells exposed to 6-OHDA or morphine. Scale bars: 10 μm. (B) Protein levels of LC3, p62 and Actin in SH-SY5Y cells treated with morphine alone for 24 h were analyzed (upper panel) and quantified (lower panel) by western blot. ∗ P
Figure Legend Snippet: The role of autophagy in morphine-induced neuroprotection in SH-SY5Y cells. (A) Double immunofluorescent staining revealed accumulation of LC3 punctuated foci in cells exposed to 6-OHDA or morphine. Scale bars: 10 μm. (B) Protein levels of LC3, p62 and Actin in SH-SY5Y cells treated with morphine alone for 24 h were analyzed (upper panel) and quantified (lower panel) by western blot. ∗ P

Techniques Used: Staining, Western Blot

Morphine suppressed 6-OHDA-induced ER stress through activation of UPR. (A,B) Morphine induced UPR in SH-SY5Y cells. Protein levels of GRP78, p-IRE1α, IRE1α, p-PERK, PERK, ATF6, CHOP and Tubulin in SH-SY5Y cells were analyzed (A) and quantified (B) by western blot. ∗ P
Figure Legend Snippet: Morphine suppressed 6-OHDA-induced ER stress through activation of UPR. (A,B) Morphine induced UPR in SH-SY5Y cells. Protein levels of GRP78, p-IRE1α, IRE1α, p-PERK, PERK, ATF6, CHOP and Tubulin in SH-SY5Y cells were analyzed (A) and quantified (B) by western blot. ∗ P

Techniques Used: Activation Assay, Western Blot

Morphine reduced the loss of dopaminergic neurons, improved motor dysfunction and inhibited pain hypersensitivity in 6-OHDA-lesioned rats. (A) Left panel is immunofluorescence of TH (green) in the SNc. Right panel is quantification of TH + nerve fibers. Scale bars: 500 μm (upper panel) and 100 μm (lower panel). (B) TH expression in SNc was analyzed (left panel) and semi-quantitatively determined (right panel) by western blotting. ∗ P
Figure Legend Snippet: Morphine reduced the loss of dopaminergic neurons, improved motor dysfunction and inhibited pain hypersensitivity in 6-OHDA-lesioned rats. (A) Left panel is immunofluorescence of TH (green) in the SNc. Right panel is quantification of TH + nerve fibers. Scale bars: 500 μm (upper panel) and 100 μm (lower panel). (B) TH expression in SNc was analyzed (left panel) and semi-quantitatively determined (right panel) by western blotting. ∗ P

Techniques Used: Immunofluorescence, Expressing, Western Blot

Morphine protected SH-SY5Y human neuroblastoma cells against 6-OHDA–induced cell apoptosis. (A) RT-qPCR analysis of the mRNA expression of CYP2D6 , CYP3A4 and CYP3A5 following 6-OHDA treatment. ∗∗∗ P
Figure Legend Snippet: Morphine protected SH-SY5Y human neuroblastoma cells against 6-OHDA–induced cell apoptosis. (A) RT-qPCR analysis of the mRNA expression of CYP2D6 , CYP3A4 and CYP3A5 following 6-OHDA treatment. ∗∗∗ P

Techniques Used: Quantitative RT-PCR, Expressing

12) Product Images from "Disrupted Dopamine Transmission and the Emergence of Exaggerated Beta Oscillations in Subthalamic Nucleus and Cerebral Cortex"

Article Title: Disrupted Dopamine Transmission and the Emergence of Exaggerated Beta Oscillations in Subthalamic Nucleus and Cerebral Cortex

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0123-08.2008

Exaggerated beta oscillations are present in the cortex and subthalamic nucleus of 6-OHDA-lesioned anesthetized rats. A , Frontal ECoGs and time-evolving power spectrogram of ECoG activity. Exaggerated beta oscillations emerge during spontaneous activation (2) but not SWA (1). Calibration: 0.2 mV, 500 ms. a.u., Arbitrary units. B , Spectrograms of simultaneously recorded activity in cortex (ECoG) and subthalamic nucleus (STN-LFP) during spontaneous activation. Weak power at 50 Hz is line noise. C , Power spectra (Power) of ECoGs, as well as LFPs simultaneously recorded from within, immediately above and below the STN, in a representative lesioned and control animal during activation. Gray boxes indicate the classic beta band (15–30 Hz). D , E , Beta oscillations are evident in the discharges of single STN units, as shown in autocorrelograms (AC) and periodograms (Lomb). The dashed line in periodogram indicates p = 0.05. STN neurons fire either spike bursts or single spikes in time with most (but not all; arrowhead in E ) beta cycles in oscillatory LFPs, as shown in spike-triggered LFP averages (STAv). Power spectra of ECoG and STN-LFP simultaneously recorded with neuron in D are also shown. Calibration: D , 0.2 mV (ECoG), 0.5 mV (unit), 0.1 mV (LFP), 100 ms; E , 0.125 mV, 100 ms. F , Beta oscillations are also reflected in rhythmic multiunit activity in STN (contacts 2–4), but not immediately above or below (contacts 1 and 5). Traces 1–5 of units and LFPs were recorded simultaneously. Calibration: 0.1 mV, 100 ms. G , Plot of STN neurons with significant oscillations in their spike trains (those recorded with silicon probes or glass electrodes are represented by diamonds and circles, respectively). H , Coherence (left) and total beta coherence at 17–23 Hz (right) between single units and LFPs in the STN as recorded with probes ( n = 14 and 8 unit/LFP pairs in lesioned and control, respectively). The dashed line is the 95% confidence limit. Data are means ±1 SEM. * p = 0.005. I , Mean firing rates of STN neurons during activated brain state. Data are means ±1 SEM. * p
Figure Legend Snippet: Exaggerated beta oscillations are present in the cortex and subthalamic nucleus of 6-OHDA-lesioned anesthetized rats. A , Frontal ECoGs and time-evolving power spectrogram of ECoG activity. Exaggerated beta oscillations emerge during spontaneous activation (2) but not SWA (1). Calibration: 0.2 mV, 500 ms. a.u., Arbitrary units. B , Spectrograms of simultaneously recorded activity in cortex (ECoG) and subthalamic nucleus (STN-LFP) during spontaneous activation. Weak power at 50 Hz is line noise. C , Power spectra (Power) of ECoGs, as well as LFPs simultaneously recorded from within, immediately above and below the STN, in a representative lesioned and control animal during activation. Gray boxes indicate the classic beta band (15–30 Hz). D , E , Beta oscillations are evident in the discharges of single STN units, as shown in autocorrelograms (AC) and periodograms (Lomb). The dashed line in periodogram indicates p = 0.05. STN neurons fire either spike bursts or single spikes in time with most (but not all; arrowhead in E ) beta cycles in oscillatory LFPs, as shown in spike-triggered LFP averages (STAv). Power spectra of ECoG and STN-LFP simultaneously recorded with neuron in D are also shown. Calibration: D , 0.2 mV (ECoG), 0.5 mV (unit), 0.1 mV (LFP), 100 ms; E , 0.125 mV, 100 ms. F , Beta oscillations are also reflected in rhythmic multiunit activity in STN (contacts 2–4), but not immediately above or below (contacts 1 and 5). Traces 1–5 of units and LFPs were recorded simultaneously. Calibration: 0.1 mV, 100 ms. G , Plot of STN neurons with significant oscillations in their spike trains (those recorded with silicon probes or glass electrodes are represented by diamonds and circles, respectively). H , Coherence (left) and total beta coherence at 17–23 Hz (right) between single units and LFPs in the STN as recorded with probes ( n = 14 and 8 unit/LFP pairs in lesioned and control, respectively). The dashed line is the 95% confidence limit. Data are means ±1 SEM. * p = 0.005. I , Mean firing rates of STN neurons during activated brain state. Data are means ±1 SEM. * p

Techniques Used: Activity Assay, Activation Assay, Mass Spectrometry

Chronic 6-OHDA lesions, but not acute antagonist treatment, exaggerate beta oscillations in the subthalamic nucleus. A , Average power spectra of STN-LFPs recorded in untreated control animals ( n = 8), controls after acute antagonist treatment ( n = 8, “treated controls”), and chronically lesioned animals ( n = 9) during the activated brain state. The gray boxes indicate the peak beta band (17–23 Hz) used in quantitative analyses in C and D . Spectral power at ∼50 Hz (line noise) was removed for clarity. a.u., Arbitrary units. B , Average power spectra of STN-LFPs recorded in the same animals during slow-wave activity. There are no distinct peaks in power at beta frequencies. C , D , Average log power of peak beta oscillations (17–23 Hz) in STN-LFPs across experimental groups and brain states. Power values in A and B were normalized by logarithmic transformation before statistical testing. Data are means ±1 SEM. * p
Figure Legend Snippet: Chronic 6-OHDA lesions, but not acute antagonist treatment, exaggerate beta oscillations in the subthalamic nucleus. A , Average power spectra of STN-LFPs recorded in untreated control animals ( n = 8), controls after acute antagonist treatment ( n = 8, “treated controls”), and chronically lesioned animals ( n = 9) during the activated brain state. The gray boxes indicate the peak beta band (17–23 Hz) used in quantitative analyses in C and D . Spectral power at ∼50 Hz (line noise) was removed for clarity. a.u., Arbitrary units. B , Average power spectra of STN-LFPs recorded in the same animals during slow-wave activity. There are no distinct peaks in power at beta frequencies. C , D , Average log power of peak beta oscillations (17–23 Hz) in STN-LFPs across experimental groups and brain states. Power values in A and B were normalized by logarithmic transformation before statistical testing. Data are means ±1 SEM. * p

Techniques Used: Activity Assay, Transformation Assay

Chronic 6-OHDA lesions, but not acute antagonist treatment, exaggerate beta oscillations in the cerebral cortex. A , Average power spectra of ECoGs recorded in untreated control animals ( n = 8), controls after acute antagonist treatment ( n = 8, “treated controls”), and chronically lesioned animals ( n = 9) during the activated brain state. The gray boxes indicate the peak beta band (17–23 Hz) used in quantitative analyses in C and D . Spectral power at ∼50 Hz (line noise) was removed for clarity. a.u., Arbitrary units. B , Average power spectra of ECoGs recorded in same animals during slow-wave activity. C , D , Average log power of peak beta oscillations (17–23 Hz) in ECoGs across experimental groups and brain states. The power values in A and B were normalized by logarithmic transformation before statistical testing. Data are means ±1 SEM. * p
Figure Legend Snippet: Chronic 6-OHDA lesions, but not acute antagonist treatment, exaggerate beta oscillations in the cerebral cortex. A , Average power spectra of ECoGs recorded in untreated control animals ( n = 8), controls after acute antagonist treatment ( n = 8, “treated controls”), and chronically lesioned animals ( n = 9) during the activated brain state. The gray boxes indicate the peak beta band (17–23 Hz) used in quantitative analyses in C and D . Spectral power at ∼50 Hz (line noise) was removed for clarity. a.u., Arbitrary units. B , Average power spectra of ECoGs recorded in same animals during slow-wave activity. C , D , Average log power of peak beta oscillations (17–23 Hz) in ECoGs across experimental groups and brain states. The power values in A and B were normalized by logarithmic transformation before statistical testing. Data are means ±1 SEM. * p

Techniques Used: Activity Assay, Transformation Assay

Temporal coupling and mutual information of multiunit activities in the subthalamic nucleus increase after 6-OHDA lesions. A , Average coherence spectra of STN multiunit activities recorded in untreated control animals ( n = 21 contact pairs), controls after acute antagonist treatment ( n = 21 contact pairs, “treated controls”), and chronically lesioned animals ( n = 23 contact pairs). The gray box indicates the beta band (17–23 Hz) used in quantitative analyses. B , Average coherence of STN activities at beta frequencies (17–23 Hz) across experimental groups. Data are means ±1 SEM. * p
Figure Legend Snippet: Temporal coupling and mutual information of multiunit activities in the subthalamic nucleus increase after 6-OHDA lesions. A , Average coherence spectra of STN multiunit activities recorded in untreated control animals ( n = 21 contact pairs), controls after acute antagonist treatment ( n = 21 contact pairs, “treated controls”), and chronically lesioned animals ( n = 23 contact pairs). The gray box indicates the beta band (17–23 Hz) used in quantitative analyses. B , Average coherence of STN activities at beta frequencies (17–23 Hz) across experimental groups. Data are means ±1 SEM. * p

Techniques Used:

Cortical beta oscillations are not augmented by acute antagonist treatments in behaving control animals, and pathological beta oscillations are delayed after 6-OHDA injections in lesioned animals. A , Right frontal ECoGs recorded in a representative dopamine-intact rat before (control) and after systemic administration of vehicle (saline) or a combination of selective D 1 and D 2 receptor antagonists (SCH-23390 at 0.5 mg/kg and raclopride at 2 mg/kg). Recording times after injections are indicated. Calibration: 0.5 mV, 250 ms. B , Power spectra of ECoGs recorded from the single animal in A , and average power spectra for the group of dopamine-intact rats ( n = 5). Recording times after injections are indicated in parentheses (in minutes). The gray boxes and dashed lines indicate the peak beta band (22–28 Hz) and wider beta band (19–31 Hz), respectively, as used in quantitative analyses in C and D . Note that antagonist treatment did not augment beta oscillations. C , Total power of peak beta oscillations (22–28 Hz, gray) and wider beta oscillations (19–31 Hz, black) averaged across all dopamine-intact rats and treatments. Data are means ±1 SEM. Animals were fully cataleptic 10–15 min after administration of D 1 /D 2 antagonists. D , Left, Average power spectra of ECoGs recorded ipsilateral to 6-OHDA lesions in behaving rats ( n = 5) on postoperative days 1, 4, and 15. Right, Comparison of peak beta power (22–28 Hz) in ECoGs recorded from lesioned, behaving rats. Data are medians (black lines) and 25th/75th percentiles (boxes). * p
Figure Legend Snippet: Cortical beta oscillations are not augmented by acute antagonist treatments in behaving control animals, and pathological beta oscillations are delayed after 6-OHDA injections in lesioned animals. A , Right frontal ECoGs recorded in a representative dopamine-intact rat before (control) and after systemic administration of vehicle (saline) or a combination of selective D 1 and D 2 receptor antagonists (SCH-23390 at 0.5 mg/kg and raclopride at 2 mg/kg). Recording times after injections are indicated. Calibration: 0.5 mV, 250 ms. B , Power spectra of ECoGs recorded from the single animal in A , and average power spectra for the group of dopamine-intact rats ( n = 5). Recording times after injections are indicated in parentheses (in minutes). The gray boxes and dashed lines indicate the peak beta band (22–28 Hz) and wider beta band (19–31 Hz), respectively, as used in quantitative analyses in C and D . Note that antagonist treatment did not augment beta oscillations. C , Total power of peak beta oscillations (22–28 Hz, gray) and wider beta oscillations (19–31 Hz, black) averaged across all dopamine-intact rats and treatments. Data are means ±1 SEM. Animals were fully cataleptic 10–15 min after administration of D 1 /D 2 antagonists. D , Left, Average power spectra of ECoGs recorded ipsilateral to 6-OHDA lesions in behaving rats ( n = 5) on postoperative days 1, 4, and 15. Right, Comparison of peak beta power (22–28 Hz) in ECoGs recorded from lesioned, behaving rats. Data are medians (black lines) and 25th/75th percentiles (boxes). * p

Techniques Used: Mass Spectrometry

13) Product Images from "Impairment of Atg5-Dependent Autophagic Flux Promotes Paraquat- and MPP+-Induced Apoptosis But Not Rotenone or 6-Hydroxydopamine Toxicity"

Article Title: Impairment of Atg5-Dependent Autophagic Flux Promotes Paraquat- and MPP+-Induced Apoptosis But Not Rotenone or 6-Hydroxydopamine Toxicity

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kft188

Caspases mediate cell death induced by the PQ, MPP + , rotenone, and 6-OHDA, whereas lysosomal hydrolase activity only regulates PQ and MPP + toxicity. Cells were pretreated with the caspase-3 inhibitor Ac-DMQD-CHO (A), the pan-caspase inhibitor Z-VAD-FMK
Figure Legend Snippet: Caspases mediate cell death induced by the PQ, MPP + , rotenone, and 6-OHDA, whereas lysosomal hydrolase activity only regulates PQ and MPP + toxicity. Cells were pretreated with the caspase-3 inhibitor Ac-DMQD-CHO (A), the pan-caspase inhibitor Z-VAD-FMK

Techniques Used: Activity Assay

Rotenone and 6-OHDA toxicity is not affected by pharmacological modulators of autophagy, or impairment of Atg5-dependent pathway. In (A, B, E, and F), cells were subjected to 1h pretreatment with rapamycin (5μM) or wortmannin (200nM). In (C, D,
Figure Legend Snippet: Rotenone and 6-OHDA toxicity is not affected by pharmacological modulators of autophagy, or impairment of Atg5-dependent pathway. In (A, B, E, and F), cells were subjected to 1h pretreatment with rapamycin (5μM) or wortmannin (200nM). In (C, D,

Techniques Used:

Putative targets for neurotoxicants and pharmacological modulators of autophagy. The parkinsonian mimetics PQ, the mitochondrial complex I inhibitors MPP + and rotenone, and the dopamine analog 6-OHDA are used in vitro and in vivo as experimental PD models.
Figure Legend Snippet: Putative targets for neurotoxicants and pharmacological modulators of autophagy. The parkinsonian mimetics PQ, the mitochondrial complex I inhibitors MPP + and rotenone, and the dopamine analog 6-OHDA are used in vitro and in vivo as experimental PD models.

Techniques Used: In Vitro, In Vivo

14) Product Images from "Oxidation of Survival Factor MEF2D in Neuronal Death and Parkinson's Disease"

Article Title: Oxidation of Survival Factor MEF2D in Neuronal Death and Parkinson's Disease

Journal: Antioxidants & Redox Signaling

doi: 10.1089/ars.2013.5399

Inhibition of MEF2D functioning after 6-OHDA-induced toxicity in the SN4741 cells. (A) Time- and dose-dependent inhibition of MEF2 transactivation activity by 6-OHDA. The effects of 6-OHDA at indicated doses (24 h, left panel ) or time points (20 μ
Figure Legend Snippet: Inhibition of MEF2D functioning after 6-OHDA-induced toxicity in the SN4741 cells. (A) Time- and dose-dependent inhibition of MEF2 transactivation activity by 6-OHDA. The effects of 6-OHDA at indicated doses (24 h, left panel ) or time points (20 μ

Techniques Used: Inhibition, Activity Assay

Oxidation of MEF2D in the 6-OHDA model of PD and in the postmortem brains of PD patients. (A) Increased levels of carbonyl MEF2D occurred in the brains of the 6-OHDA-treated mice. The lysates prepared from the SNc region of mice 3 days after a unilateral
Figure Legend Snippet: Oxidation of MEF2D in the 6-OHDA model of PD and in the postmortem brains of PD patients. (A) Increased levels of carbonyl MEF2D occurred in the brains of the 6-OHDA-treated mice. The lysates prepared from the SNc region of mice 3 days after a unilateral

Techniques Used: Mouse Assay

Degradation of MEF2D by a lysosome-mediated pathway after oxidative stress in the SN4741 cells. (A) Attenuation of 6-OHDA-induced degradation of MEF2D by NH 4 Cl. The SN4741 cells were exposed to 6-OHDA with or without NH 4 Cl for 12 h, and cytoplasmic
Figure Legend Snippet: Degradation of MEF2D by a lysosome-mediated pathway after oxidative stress in the SN4741 cells. (A) Attenuation of 6-OHDA-induced degradation of MEF2D by NH 4 Cl. The SN4741 cells were exposed to 6-OHDA with or without NH 4 Cl for 12 h, and cytoplasmic

Techniques Used:

Oxidative modifications of MEF2D after 6-OHDA treatment. (A) Levels of MEF2D in SN4741 cells after short-term ( left panel : 20 or 40 μ M for 18 h) and long-term ( right panel : 20 or 40 μ M for 30 h) exposure
Figure Legend Snippet: Oxidative modifications of MEF2D after 6-OHDA treatment. (A) Levels of MEF2D in SN4741 cells after short-term ( left panel : 20 or 40 μ M for 18 h) and long-term ( right panel : 20 or 40 μ M for 30 h) exposure

Techniques Used:

Activation of CMA by 6-OHDA. (A) Increased levels of LAMP2A mRNA after 6-OHDA treatment. Levels of LAMP2A mRNA in the total RNA isolated from the SN4741 cells after 6-OHDA treatment (4 h) were analyzed using quantitative RT-PCR. The relative values
Figure Legend Snippet: Activation of CMA by 6-OHDA. (A) Increased levels of LAMP2A mRNA after 6-OHDA treatment. Levels of LAMP2A mRNA in the total RNA isolated from the SN4741 cells after 6-OHDA treatment (4 h) were analyzed using quantitative RT-PCR. The relative values

Techniques Used: Activation Assay, Isolation, Quantitative RT-PCR

Increased binding of oxidized MEF2D to Hsc70 and its uptake by lysosomes. (A) Increased levels of carbonyl MEF2D after 6-OHDA treatment after inhibition of lysosomal hydrolase activities. The lysates from the SN4741 cells after 18 h of 6-OHDA
Figure Legend Snippet: Increased binding of oxidized MEF2D to Hsc70 and its uptake by lysosomes. (A) Increased levels of carbonyl MEF2D after 6-OHDA treatment after inhibition of lysosomal hydrolase activities. The lysates from the SN4741 cells after 18 h of 6-OHDA

Techniques Used: Binding Assay, Inhibition

15) Product Images from "Characteristic response of striatal astrocytes to dopamine depletion"

Article Title: Characteristic response of striatal astrocytes to dopamine depletion

Journal: Neural Regeneration Research

doi: 10.4103/1673-5374.266917

Immunohistochemistry of S100B and STAT3 in the striatum following dopamine depletion. (A, A’, C, C’) Control group; (B, B’, D, D’) 6-OHDA group. (A’–D’) Enlargement of the boxed region of A, B, C, and D, respectively. S100B-immunoreactive cells were uniformly distributed, with small somas and few processes (A, B). STAT3-immunoreactive cells were uniformly distributed in the striatum, and cell bodies were small with few processes (C’, D’). Some of the larger STAT3-immunoreactive cells were observed to be interneurons, as determined by morphological characteristics of striatal cells (C’, D’). Scale bars: 90 µm in A–D; 30 µm in A’ and B’; 45 µm in C’ and D’. 6-OHDA: 6-Hydroxydopamine; Ctrl: control; S100B: calcium-binding protein B; STAT3: signal transducer and activator of transcription 3.
Figure Legend Snippet: Immunohistochemistry of S100B and STAT3 in the striatum following dopamine depletion. (A, A’, C, C’) Control group; (B, B’, D, D’) 6-OHDA group. (A’–D’) Enlargement of the boxed region of A, B, C, and D, respectively. S100B-immunoreactive cells were uniformly distributed, with small somas and few processes (A, B). STAT3-immunoreactive cells were uniformly distributed in the striatum, and cell bodies were small with few processes (C’, D’). Some of the larger STAT3-immunoreactive cells were observed to be interneurons, as determined by morphological characteristics of striatal cells (C’, D’). Scale bars: 90 µm in A–D; 30 µm in A’ and B’; 45 µm in C’ and D’. 6-OHDA: 6-Hydroxydopamine; Ctrl: control; S100B: calcium-binding protein B; STAT3: signal transducer and activator of transcription 3.

Techniques Used: Immunohistochemistry, Binding Assay

Effect of 6-OHDA on astrocytes in the striatum (immunohistochemical staining). (A, A’) Control group; (B, B’) 6-OHDA group. (A’, B’) Enlargements of the * region of A and B, respectively. GFAP-immunoreactive cells displayed intense staining, thickened processes, and enlarged somas in the 6-OHDA group. Scale bars: 120 µm in A, B; 60 µm in A’, B’. 6-OHDA: 6-Hydroxydopamine; Ctrl: control; GFAP: glial fibrillary astrocytic protein.
Figure Legend Snippet: Effect of 6-OHDA on astrocytes in the striatum (immunohistochemical staining). (A, A’) Control group; (B, B’) 6-OHDA group. (A’, B’) Enlargements of the * region of A and B, respectively. GFAP-immunoreactive cells displayed intense staining, thickened processes, and enlarged somas in the 6-OHDA group. Scale bars: 120 µm in A, B; 60 µm in A’, B’. 6-OHDA: 6-Hydroxydopamine; Ctrl: control; GFAP: glial fibrillary astrocytic protein.

Techniques Used: Immunohistochemistry, Staining

Effect of striatal dopamine depletion on GFAP, S100B, and STAT3 protein expression levels. (A) S100B- and STAT3-immunoreactive cells were increased in the 6-OHDA group compared with the control group. (B) Quantitative results of immunofluorescence double-labeling of GFAP/S100B and GFAP/STAT3, which indicate that the number of both GFAP/S100B and GFAP/STAT3 coexpressing cells increased in the 6-OHDA group compared with the control group. (C) Protein levels of GFAP, S100B, and STAT3 in the striatum; GFAP, S10B and STAT3 protein levels were increased in the 6-OHDA group compared with the control group. * P
Figure Legend Snippet: Effect of striatal dopamine depletion on GFAP, S100B, and STAT3 protein expression levels. (A) S100B- and STAT3-immunoreactive cells were increased in the 6-OHDA group compared with the control group. (B) Quantitative results of immunofluorescence double-labeling of GFAP/S100B and GFAP/STAT3, which indicate that the number of both GFAP/S100B and GFAP/STAT3 coexpressing cells increased in the 6-OHDA group compared with the control group. (C) Protein levels of GFAP, S100B, and STAT3 in the striatum; GFAP, S10B and STAT3 protein levels were increased in the 6-OHDA group compared with the control group. * P

Techniques Used: Expressing, Immunofluorescence, Labeling

Coexpression of striatal astrocytes with S100B and STAT3 following dopamine depletion. (A–D) GFAP-immunoreactive cells (green); (A’, B’) S100B-immunoreactive cells (red); (C’, D’) STAT3-immunoreactive cells (red); (A*, B*) the merged images (GFAP/S100B, yellow); (C*, D*) the merged images (GFAP/STAT3, yellow). (A, A’, C, C’) Control group; (B, B’, D, D’) 6-OHDA group. The number of cells coexpressing GFAP/S100B was higher in the 6-OHDA group (B*) than in the control group (A*). The number of GFAP/STAT3 double-labeled cells was also significantly increased in the 6-OHDA group (D*) compared with the control group (C*). Scale bar: 50 µm. 6-OHDA: 6-Hydroxydopamine; Ctrl: control; GFAP: glial fibrillary astrocytic protein; S100B: calcium-binding protein B; STAT3: signal transducer and activator of transcription 3.
Figure Legend Snippet: Coexpression of striatal astrocytes with S100B and STAT3 following dopamine depletion. (A–D) GFAP-immunoreactive cells (green); (A’, B’) S100B-immunoreactive cells (red); (C’, D’) STAT3-immunoreactive cells (red); (A*, B*) the merged images (GFAP/S100B, yellow); (C*, D*) the merged images (GFAP/STAT3, yellow). (A, A’, C, C’) Control group; (B, B’, D, D’) 6-OHDA group. The number of cells coexpressing GFAP/S100B was higher in the 6-OHDA group (B*) than in the control group (A*). The number of GFAP/STAT3 double-labeled cells was also significantly increased in the 6-OHDA group (D*) compared with the control group (C*). Scale bar: 50 µm. 6-OHDA: 6-Hydroxydopamine; Ctrl: control; GFAP: glial fibrillary astrocytic protein; S100B: calcium-binding protein B; STAT3: signal transducer and activator of transcription 3.

Techniques Used: Labeling, Binding Assay

16) Product Images from "Lower Affinity of Isradipine for L-Type Ca2+ Channels during Substantia Nigra Dopamine Neuron-Like Activity: Implications for Neuroprotection in Parkinson's Disease"

Article Title: Lower Affinity of Isradipine for L-Type Ca2+ Channels during Substantia Nigra Dopamine Neuron-Like Activity: Implications for Neuroprotection in Parkinson's Disease

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2946-16.2017

Evaluation of neuroprotective effects by Ca v 1.3 knockout or in vivo ISR pretreatment in a 6-OHDA PD mouse model (using male mice). A , B , Immunohistochemical stainings of TH + SN DA neurons in rostral sections of wild-type and Ca v 1.3 −/− mice ( A ) or wild-type mice ( B ) treated with ISR (6 or 9 mg/kg) or placebo 7 d before unilateral injection with 4.1 μg of 6-OHDA. Scale bars, 100 μm. C , D , The percentage of neurons remaining in the injected side compared with the noninjected side for each mouse were calculated 28 d after lesioning. Data are reported as the mean ± SEM. E , Distribution of ISR plasma levels in mice 35 d after pellet implantation with ISR plasma concentrations ≥3 ng/ml (mean ± SEM: 5.5 ± 0.5 ng/ml). F , A statistically significant difference for the absolute numbers of TH + cells in the nonlesioned side was only observed between wild-type (WT) and Ca v 1.3 −/− mice (wild-type mice: 6572 ± 285, n = 23; Ca v 1.3 −/− mice: 5668 ± 282, n = 27; four independent experiments; p = 0.03; unpaired Student's t test) but not between ISR- or placebo-treated wild-type mice (ISR: 6655 ± 398, n = 18; placebo: 6861 ± 381, n = 29; four independent experiments). *** p
Figure Legend Snippet: Evaluation of neuroprotective effects by Ca v 1.3 knockout or in vivo ISR pretreatment in a 6-OHDA PD mouse model (using male mice). A , B , Immunohistochemical stainings of TH + SN DA neurons in rostral sections of wild-type and Ca v 1.3 −/− mice ( A ) or wild-type mice ( B ) treated with ISR (6 or 9 mg/kg) or placebo 7 d before unilateral injection with 4.1 μg of 6-OHDA. Scale bars, 100 μm. C , D , The percentage of neurons remaining in the injected side compared with the noninjected side for each mouse were calculated 28 d after lesioning. Data are reported as the mean ± SEM. E , Distribution of ISR plasma levels in mice 35 d after pellet implantation with ISR plasma concentrations ≥3 ng/ml (mean ± SEM: 5.5 ± 0.5 ng/ml). F , A statistically significant difference for the absolute numbers of TH + cells in the nonlesioned side was only observed between wild-type (WT) and Ca v 1.3 −/− mice (wild-type mice: 6572 ± 285, n = 23; Ca v 1.3 −/− mice: 5668 ± 282, n = 27; four independent experiments; p = 0.03; unpaired Student's t test) but not between ISR- or placebo-treated wild-type mice (ISR: 6655 ± 398, n = 18; placebo: 6861 ± 381, n = 29; four independent experiments). *** p

Techniques Used: Knock-Out, In Vivo, Mouse Assay, Immunohistochemistry, Injection

17) Product Images from "Achyranthes bidentata polypeptide protects dopaminergic neurons from apoptosis induced by rotenone and 6-hydroxydopamine"

Article Title: Achyranthes bidentata polypeptide protects dopaminergic neurons from apoptosis induced by rotenone and 6-hydroxydopamine

Journal: Neural Regeneration Research

doi: 10.4103/1673-5374.239446

ABPPk attenuated LDH activity measured by a microplate reader in 6-OHDA- or rotenone-treated cells. (A, B) SH-SY5Y cells were pretreated with different concentrations of ABPPk for 12 hours, followed by 150 μM 6-OHDA (A) or 200 μM rotenone (B) for an additional 36 hours. (C, D) Primary rat DNs were pretreated with 50 ng/mL of ABPPk and then 50 μM 6-OHDA (C) or 50 μM rotenone (D). Data are presented as the mean ± SEM (one-way analysis of variance followed by Bonferroni post hoc test). *** P
Figure Legend Snippet: ABPPk attenuated LDH activity measured by a microplate reader in 6-OHDA- or rotenone-treated cells. (A, B) SH-SY5Y cells were pretreated with different concentrations of ABPPk for 12 hours, followed by 150 μM 6-OHDA (A) or 200 μM rotenone (B) for an additional 36 hours. (C, D) Primary rat DNs were pretreated with 50 ng/mL of ABPPk and then 50 μM 6-OHDA (C) or 50 μM rotenone (D). Data are presented as the mean ± SEM (one-way analysis of variance followed by Bonferroni post hoc test). *** P

Techniques Used: Activity Assay

ABPPk increases Bcl-2/Bax ratio detected by western blot assay in injured cells. (A, B) SH-SY5Y cells were pretreated with 50 ng/mL of ABPPk for 12 hours, followed by 150 μM 6-OHDA (A) or 200 μM rotenone (B) for an additional 36 hours to induce cell apoptosis. Protein levels of Bcl-2 and Bax were measured by western blot assay with the antibodies as indicated. (C, D) Primary rat dopaminergic neurons were pretreated with 50 ng/mL of ABPPk, followed by 50 μM 6-OHDA (C) or 50 μM rotenone (D). Protein levels of Bcl-2 and Bax were measured by western blot assay with the antibodies as indicated. Actin was used as a loading control. Data are presented as the mean ± SEM (one-way analysis of variance followed by Bonferroni post hoc test). ** P
Figure Legend Snippet: ABPPk increases Bcl-2/Bax ratio detected by western blot assay in injured cells. (A, B) SH-SY5Y cells were pretreated with 50 ng/mL of ABPPk for 12 hours, followed by 150 μM 6-OHDA (A) or 200 μM rotenone (B) for an additional 36 hours to induce cell apoptosis. Protein levels of Bcl-2 and Bax were measured by western blot assay with the antibodies as indicated. (C, D) Primary rat dopaminergic neurons were pretreated with 50 ng/mL of ABPPk, followed by 50 μM 6-OHDA (C) or 50 μM rotenone (D). Protein levels of Bcl-2 and Bax were measured by western blot assay with the antibodies as indicated. Actin was used as a loading control. Data are presented as the mean ± SEM (one-way analysis of variance followed by Bonferroni post hoc test). ** P

Techniques Used: Western Blot

ABPPk improved cell viability detected by MTT assay in the presence of 6-OHDA and rotenone. (A, B) SH-SY5Y cells were pretreated with different concentrations of ABPPk for 12 hours, followed by 150 μM 6-OHDA (A) or 200 μM rotenone (B) for an additional 36 hours. (C, D) Primary rat DNs were pretreated with 50 ng/mL of ABPPk and then 50 μM 6-OHDA (C) or 50 μM rotenone (D). Data are presented as the mean ± SEM (one-way analysis of variance followed by Bonferroni post hoc test). *** P
Figure Legend Snippet: ABPPk improved cell viability detected by MTT assay in the presence of 6-OHDA and rotenone. (A, B) SH-SY5Y cells were pretreated with different concentrations of ABPPk for 12 hours, followed by 150 μM 6-OHDA (A) or 200 μM rotenone (B) for an additional 36 hours. (C, D) Primary rat DNs were pretreated with 50 ng/mL of ABPPk and then 50 μM 6-OHDA (C) or 50 μM rotenone (D). Data are presented as the mean ± SEM (one-way analysis of variance followed by Bonferroni post hoc test). *** P

Techniques Used: MTT Assay

TUNEL assays showing cell apoptosis in SH-SY5Y cells and primary dopaminergic neurons following ABPPk treatment. (A, B) SH-SY5Y cells were pretreated with 50 ng/mL of ABPPk for 12 hours, followed by 150 μM 6-OHDA (A) or 200 μM rotenone (B) for an additional 36 hours. (C, D) Primary DNs were pretreated with 50 ng/mL of ABPPk for 12 hours, followed by 50 μM 6-OHDA (C) or 50 μM rotenone (D) for an additional 36 hours. Cell viability was measured using the TUNEL assay. TUNEL-positive cells (red) indicate apoptotic cells. Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) was used to stain the nucleus (blue). Scale bars: 50 μm. Data are presented as the mean ± SEM (one-way analysis of variance followed by Bonferroni post hoc test). *** P
Figure Legend Snippet: TUNEL assays showing cell apoptosis in SH-SY5Y cells and primary dopaminergic neurons following ABPPk treatment. (A, B) SH-SY5Y cells were pretreated with 50 ng/mL of ABPPk for 12 hours, followed by 150 μM 6-OHDA (A) or 200 μM rotenone (B) for an additional 36 hours. (C, D) Primary DNs were pretreated with 50 ng/mL of ABPPk for 12 hours, followed by 50 μM 6-OHDA (C) or 50 μM rotenone (D) for an additional 36 hours. Cell viability was measured using the TUNEL assay. TUNEL-positive cells (red) indicate apoptotic cells. Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) was used to stain the nucleus (blue). Scale bars: 50 μm. Data are presented as the mean ± SEM (one-way analysis of variance followed by Bonferroni post hoc test). *** P

Techniques Used: TUNEL Assay, Staining

ABPPk improved mitochondrial membrane potential in SH-SH5Y cells and primary dopaminergic neurons. (A, B) SH-SY5Y cells were pretreated with 50 ng/mL of ABPPk for 12 hours, followed by 150 μM 6-OHDA (A) or 200 μM rotenone (B) for an additional 36 hours. (C, D) Primary dopaminergic neurons were pretreated with 50 ng/mL of ABPPk for 12 hours, followed by 50 μM 6-OHDA (C) or 50 μM rotenone (D) for an additional 36 hours. Mitochondrial staining was performed using a mitochondrial-specific Cytopainter (ab112145; Abcam, Cambridge, MA, USA), and quantified using National Institutes of Health (NIH) ImageJ software (Bethesda, MD, USA). Scale bars: 50 μm. Data are presented as the mean ± SEM (one-way analysis of variance followed by Bonferroni post hoc test). ** P
Figure Legend Snippet: ABPPk improved mitochondrial membrane potential in SH-SH5Y cells and primary dopaminergic neurons. (A, B) SH-SY5Y cells were pretreated with 50 ng/mL of ABPPk for 12 hours, followed by 150 μM 6-OHDA (A) or 200 μM rotenone (B) for an additional 36 hours. (C, D) Primary dopaminergic neurons were pretreated with 50 ng/mL of ABPPk for 12 hours, followed by 50 μM 6-OHDA (C) or 50 μM rotenone (D) for an additional 36 hours. Mitochondrial staining was performed using a mitochondrial-specific Cytopainter (ab112145; Abcam, Cambridge, MA, USA), and quantified using National Institutes of Health (NIH) ImageJ software (Bethesda, MD, USA). Scale bars: 50 μm. Data are presented as the mean ± SEM (one-way analysis of variance followed by Bonferroni post hoc test). ** P

Techniques Used: Staining, Software

18) Product Images from "Structural Changes Observed in the Piriform Cortex in a Rat Model of Pre-motor Parkinson’s Disease"

Article Title: Structural Changes Observed in the Piriform Cortex in a Rat Model of Pre-motor Parkinson’s Disease

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2018.00479

Protocol for dual neurotoxin administration in rats to generate a pre-motor PD model. Behavioral testing (sucrose preference test, rotarod, novel object recognition, hidden food test, habituation/dishabituation test) was carried out before, after toxins and after the 7-day treatment with exendin-4 (EX-4). The study included five experimental groups: sham, sham + EX-4, 6-OHDA + DSP-4 (model), 6-OHDA + DSP-4 + EX-4 (model + EX-4) and 6-OHDA + DSP-4 + EX-4 + EX9-39 (model + EX-4 + EX9-39). Histological procedures occurred after animals were culled at days 23–24.
Figure Legend Snippet: Protocol for dual neurotoxin administration in rats to generate a pre-motor PD model. Behavioral testing (sucrose preference test, rotarod, novel object recognition, hidden food test, habituation/dishabituation test) was carried out before, after toxins and after the 7-day treatment with exendin-4 (EX-4). The study included five experimental groups: sham, sham + EX-4, 6-OHDA + DSP-4 (model), 6-OHDA + DSP-4 + EX-4 (model + EX-4) and 6-OHDA + DSP-4 + EX-4 + EX9-39 (model + EX-4 + EX9-39). Histological procedures occurred after animals were culled at days 23–24.

Techniques Used:

19) Product Images from "Modulation by Trace Amine-Associated Receptor 1 of Experimental Parkinsonism, l-DOPA Responsivity, and Glutamatergic Neurotransmission"

Article Title: Modulation by Trace Amine-Associated Receptor 1 of Experimental Parkinsonism, l-DOPA Responsivity, and Glutamatergic Neurotransmission

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1312-15.2015

Effect of TAAR1 agonism on intrastriatal 6-OHDA administration. A , Intrastriatal 6-OHDA injections caused a significant reduction of striatal DAT binding, detected by [ 125 I]RTI-55, in mice chronically administered with RO5166017 compared with vehicle-treated mice. B , DAT ratio between the lesioned and the intact hemispheres after intrastriatal 6-OHDA injections was in mice treated with RO5166017 compared with vehicle-treated mice. VEH, vehicle; RO, RO5166017. ## p
Figure Legend Snippet: Effect of TAAR1 agonism on intrastriatal 6-OHDA administration. A , Intrastriatal 6-OHDA injections caused a significant reduction of striatal DAT binding, detected by [ 125 I]RTI-55, in mice chronically administered with RO5166017 compared with vehicle-treated mice. B , DAT ratio between the lesioned and the intact hemispheres after intrastriatal 6-OHDA injections was in mice treated with RO5166017 compared with vehicle-treated mice. VEH, vehicle; RO, RO5166017. ## p

Techniques Used: Binding Assay, Mouse Assay

Levels of TH and dopamine transporter in WT and TAAR1 KO mice after intrastriatal or MFB 6-OHDA administration. A , TH levels in striatal tissue from WT and TAAR1 KO mice were reduced by striatal 6-OHDA injections. TH levels were higher in both the intact and the lesioned hemispheres of TAAR1 KO mice compared with WT mice. B , TH ratio between the lesioned and the intact hemispheres after intrastriatal 6-OHDA injections showed a higher TH ratio in TAAR1 KO mice than WT mice. C , Intrastriatal 6-OHDA injections caused a significant reduction of DAT binding, detected by [ 125 I]RTI-55, at the level of striatum in both WT and TAAR1 KO mice. DAT levels were higher in the intact and the lesioned hemispheres in TAAR1 KO mice compared with WT mice. D , DAT ratio between the lesioned and the intact hemispheres after intrastriatal 6-OHDA injections showed a higher DAT ratio in TAAR1 KO mice than WT mice. E , Striatal tissue from WT and TAAR1 KO mice with MFB 6-OHDA injections showed a near-complete reduction of TH in the lesioned side of both genotypes. F , TH ratio between the lesioned and the intact hemisphere after MFB 6-OHDA injections did not differ between WT and TAAR1 KO mice. DAT, Dopamine transporter. # p
Figure Legend Snippet: Levels of TH and dopamine transporter in WT and TAAR1 KO mice after intrastriatal or MFB 6-OHDA administration. A , TH levels in striatal tissue from WT and TAAR1 KO mice were reduced by striatal 6-OHDA injections. TH levels were higher in both the intact and the lesioned hemispheres of TAAR1 KO mice compared with WT mice. B , TH ratio between the lesioned and the intact hemispheres after intrastriatal 6-OHDA injections showed a higher TH ratio in TAAR1 KO mice than WT mice. C , Intrastriatal 6-OHDA injections caused a significant reduction of DAT binding, detected by [ 125 I]RTI-55, at the level of striatum in both WT and TAAR1 KO mice. DAT levels were higher in the intact and the lesioned hemispheres in TAAR1 KO mice compared with WT mice. D , DAT ratio between the lesioned and the intact hemispheres after intrastriatal 6-OHDA injections showed a higher DAT ratio in TAAR1 KO mice than WT mice. E , Striatal tissue from WT and TAAR1 KO mice with MFB 6-OHDA injections showed a near-complete reduction of TH in the lesioned side of both genotypes. F , TH ratio between the lesioned and the intact hemisphere after MFB 6-OHDA injections did not differ between WT and TAAR1 KO mice. DAT, Dopamine transporter. # p

Techniques Used: Mouse Assay, Binding Assay

Tonic striatal glutamate release and the reuptake rate (T80) of evoked striatal glutamate in 6-OHDA-treated and cNurr1 DATCreER KO mice. A , Tonic extracellular glutamate concentrations in striatum of MFB 6-OHDA-treated mice were not affected by lesion or ropinirole administration. B , Tonic extracellular glutamate concentrations were not significantly affected in cNurr1 DATCreER KO mice or by TAAR1 ligands. C , Local administration of AM251 significantly enhanced tonic glutamate concentrations in cNurr1 DATCreER KO mice. D , T80 was prolonged in striatum of 6-OHDA-treated mice after ejection of KCl. Local application of ropinirole inhibited this effect. E , No T80 alterations were seen in striatum of cNurr1 DATCreER KO mice compared with WT mice or mice after TAAR1 ligands, ropinirole, or AM251. Coadministration with RO5166017 attenuated the AM251-induced hyperglutamatergic state. * p
Figure Legend Snippet: Tonic striatal glutamate release and the reuptake rate (T80) of evoked striatal glutamate in 6-OHDA-treated and cNurr1 DATCreER KO mice. A , Tonic extracellular glutamate concentrations in striatum of MFB 6-OHDA-treated mice were not affected by lesion or ropinirole administration. B , Tonic extracellular glutamate concentrations were not significantly affected in cNurr1 DATCreER KO mice or by TAAR1 ligands. C , Local administration of AM251 significantly enhanced tonic glutamate concentrations in cNurr1 DATCreER KO mice. D , T80 was prolonged in striatum of 6-OHDA-treated mice after ejection of KCl. Local application of ropinirole inhibited this effect. E , No T80 alterations were seen in striatum of cNurr1 DATCreER KO mice compared with WT mice or mice after TAAR1 ligands, ropinirole, or AM251. Coadministration with RO5166017 attenuated the AM251-induced hyperglutamatergic state. * p

Techniques Used: Mouse Assay

DAT levels and striatal glutamate release under dopamine deficiency and the modulatory effects of TAAR1, ropinirole, and AM251. A , Autoradiographic detection of DAT by [ 125 I]RTI-55 in brain slices revealed a 97 ± 1.3% reduction in the lesioned compared with the intact hemisphere of MFB 6-OHDA-treated C57BL/6J mice ( n = 10). B , Real-time in vivo amperometry revealed significantly enhanced glutamate release in the lesioned hemisphere compared with the intact hemisphere. This hyperglutamatergic state was reversed after local application of the TAAR1 agonist RO5155017. C , There was a 67 ± 3.8% reduction in DAT levels in cNurr1 DATCreER KO ( n = 6) compared with WT mice ( n = 6). D , KCl-evoked glutamate concentrations were significantly enhanced in the striatum of KO mice compared with WT mice. This effect was normalized after local application of RO5166017. E , Local administration of EPPTB did not significantly affect KCl-evoked striatal glutamate release in cNurr1 DATCreER KO animals, but blocked the effect of RO5166017 when these compounds were coadministered. F , Local administration of ropinirole attenuated KCl-evoked glutamate amplitudes in striatum. Coadministration of ropinirole did not significantly change the effects of RO5166017 or EPPTB in cNurr1 DATCreER KO animals. AM251 had no effects by itself, but attenuated the inhibitory effect of RO5166017 on KCl-evoked glutamate release. Likewise, AM251 attenuated the inhibitory effect of ropinirole. The WT vehicle, cNurr1 DATCreER KO vehicle, and RO5166017 data have been included in several graphs to facilitate comparisons. Data are presented as the mean ± SEM from 4–5 glutamate peaks from 5–10 animals/treatment. DAT, Dopamine transporter;. + p
Figure Legend Snippet: DAT levels and striatal glutamate release under dopamine deficiency and the modulatory effects of TAAR1, ropinirole, and AM251. A , Autoradiographic detection of DAT by [ 125 I]RTI-55 in brain slices revealed a 97 ± 1.3% reduction in the lesioned compared with the intact hemisphere of MFB 6-OHDA-treated C57BL/6J mice ( n = 10). B , Real-time in vivo amperometry revealed significantly enhanced glutamate release in the lesioned hemisphere compared with the intact hemisphere. This hyperglutamatergic state was reversed after local application of the TAAR1 agonist RO5155017. C , There was a 67 ± 3.8% reduction in DAT levels in cNurr1 DATCreER KO ( n = 6) compared with WT mice ( n = 6). D , KCl-evoked glutamate concentrations were significantly enhanced in the striatum of KO mice compared with WT mice. This effect was normalized after local application of RO5166017. E , Local administration of EPPTB did not significantly affect KCl-evoked striatal glutamate release in cNurr1 DATCreER KO animals, but blocked the effect of RO5166017 when these compounds were coadministered. F , Local administration of ropinirole attenuated KCl-evoked glutamate amplitudes in striatum. Coadministration of ropinirole did not significantly change the effects of RO5166017 or EPPTB in cNurr1 DATCreER KO animals. AM251 had no effects by itself, but attenuated the inhibitory effect of RO5166017 on KCl-evoked glutamate release. Likewise, AM251 attenuated the inhibitory effect of ropinirole. The WT vehicle, cNurr1 DATCreER KO vehicle, and RO5166017 data have been included in several graphs to facilitate comparisons. Data are presented as the mean ± SEM from 4–5 glutamate peaks from 5–10 animals/treatment. DAT, Dopamine transporter;. + p

Techniques Used: Mouse Assay, In Vivo

20) Product Images from "The CB1 cannabinoid receptor agonist reduces L-DOPA-induced motor fluctuation and ERK1/2 phosphorylation in 6-OHDA-lesioned rats"

Article Title: The CB1 cannabinoid receptor agonist reduces L-DOPA-induced motor fluctuation and ERK1/2 phosphorylation in 6-OHDA-lesioned rats

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S60944

Effects of chronic administration of WIN-55,212-2 on dyskinetic responses to L-DOPA in 6-OHDA-lesioned rats. Notes: ( A ) Effects of WIN-55,212-2 on the duration of rotational responses to L-DOPA. ( B ) Effects of WIN-55,212-2 on peak turning responses to L-DOPA. In 6-OHDA-lesioned rats, WIN-55,212-2 (1 mg/kg, IP) or vehicle was co-administered with L-DOPA (50 mg/kg, IP) and benserazide (12.5 mg/kg, IP) for 21 days. Rotational responses and peak turning were measured immediately after L-DOPA injection at day 1, 7, 14, and 21. Data are expressed as means ± SEM (n=7 per group). * P
Figure Legend Snippet: Effects of chronic administration of WIN-55,212-2 on dyskinetic responses to L-DOPA in 6-OHDA-lesioned rats. Notes: ( A ) Effects of WIN-55,212-2 on the duration of rotational responses to L-DOPA. ( B ) Effects of WIN-55,212-2 on peak turning responses to L-DOPA. In 6-OHDA-lesioned rats, WIN-55,212-2 (1 mg/kg, IP) or vehicle was co-administered with L-DOPA (50 mg/kg, IP) and benserazide (12.5 mg/kg, IP) for 21 days. Rotational responses and peak turning were measured immediately after L-DOPA injection at day 1, 7, 14, and 21. Data are expressed as means ± SEM (n=7 per group). * P

Techniques Used: Injection

Effects of acute administration of WIN-55,212-2 on dyskinetic responses to L-DOPA in 6-OHDA-lesioned rats. Notes: ( A ) Effects of chronic L-DOPA administration on the duration of rotational responses. ( B ) Effects of WIN-55,212-2 on the duration of rotational responses to L-DOPA. ( C ) Effects of chronic L-DOPA administration on peak turning responses. ( D ) Effects of WIN-55,212-2 on peak turning responses to L-DOPA. All 6-OHDA-lesioned rats received IP injections of L-DOPA at 50 mg/kg and benserazide at 12.5 mg/kg (twice daily for 21 days). Rotational responses and peak turning were measured immediately after L-DOPA injections at day 1, 7, 14, and 21 ( A and C ). At day 22, rats were randomly divided into two groups (n=7 per group) and received co-administration of WIN-55,212-2 (1 mg/kg, IP) or vehicle with L-DOPA/benserazide. Behavioral activities were measured following L-DOPA injection ( C and D ). * P
Figure Legend Snippet: Effects of acute administration of WIN-55,212-2 on dyskinetic responses to L-DOPA in 6-OHDA-lesioned rats. Notes: ( A ) Effects of chronic L-DOPA administration on the duration of rotational responses. ( B ) Effects of WIN-55,212-2 on the duration of rotational responses to L-DOPA. ( C ) Effects of chronic L-DOPA administration on peak turning responses. ( D ) Effects of WIN-55,212-2 on peak turning responses to L-DOPA. All 6-OHDA-lesioned rats received IP injections of L-DOPA at 50 mg/kg and benserazide at 12.5 mg/kg (twice daily for 21 days). Rotational responses and peak turning were measured immediately after L-DOPA injections at day 1, 7, 14, and 21 ( A and C ). At day 22, rats were randomly divided into two groups (n=7 per group) and received co-administration of WIN-55,212-2 (1 mg/kg, IP) or vehicle with L-DOPA/benserazide. Behavioral activities were measured following L-DOPA injection ( C and D ). * P

Techniques Used: Injection

Effects of chronic administration of WIN-55,212-2 on L-DOPA-stimulated ERK1/2 phosphorylation in 6-OHDA-lesioned rats. Notes: Representative Western blots are shown above the quantification of immunoblot results. Note that WIN-55,212-2 substantially blocked L-DOPA-stimulated ERK1/2 phosphorylation. In the three groups of 6-OHDA-lesioned rats, daily injections of vehicle, vehicle plus L-DOPA, or WIN-55,212-2 plus L-DOPA were made for 21 days. At day 22, rats were sacrificed and striatal tissue was dissected for immunoblot analysis. Data area expressed as means ± SEM (n=6 per group). * P
Figure Legend Snippet: Effects of chronic administration of WIN-55,212-2 on L-DOPA-stimulated ERK1/2 phosphorylation in 6-OHDA-lesioned rats. Notes: Representative Western blots are shown above the quantification of immunoblot results. Note that WIN-55,212-2 substantially blocked L-DOPA-stimulated ERK1/2 phosphorylation. In the three groups of 6-OHDA-lesioned rats, daily injections of vehicle, vehicle plus L-DOPA, or WIN-55,212-2 plus L-DOPA were made for 21 days. At day 22, rats were sacrificed and striatal tissue was dissected for immunoblot analysis. Data area expressed as means ± SEM (n=6 per group). * P

Techniques Used: Western Blot

Effects of chronic administration of WIN-55,212-2 on L-DOPA-stimulated DARPP-32 phosphorylation in 6-OHDA-lesioned rats. Notes: Representative Western blots are shown above the quantification of immunoblot results. Note that WIN-55,212-2 substantially changed L-DOPA-stimulated DARPP-32 phosphorylation. In the three groups of 6-OHDA-lesioned rats, daily injections of vehicle, vehicle plus L-DOPA, or WIN-55,212-2 plus L-DOPA were made for 21 days. At day 22, rats were sacrificed and striatal tissue was dissected for immunoblot analysis. Data are expressed as means ± SEM (n=6 per group). * P
Figure Legend Snippet: Effects of chronic administration of WIN-55,212-2 on L-DOPA-stimulated DARPP-32 phosphorylation in 6-OHDA-lesioned rats. Notes: Representative Western blots are shown above the quantification of immunoblot results. Note that WIN-55,212-2 substantially changed L-DOPA-stimulated DARPP-32 phosphorylation. In the three groups of 6-OHDA-lesioned rats, daily injections of vehicle, vehicle plus L-DOPA, or WIN-55,212-2 plus L-DOPA were made for 21 days. At day 22, rats were sacrificed and striatal tissue was dissected for immunoblot analysis. Data are expressed as means ± SEM (n=6 per group). * P

Techniques Used: Western Blot

21) Product Images from "Mitochondrial Complex I Reversible S-Nitrosation Improves Bioenergetics and Is Protective in Parkinson's Disease"

Article Title: Mitochondrial Complex I Reversible S-Nitrosation Improves Bioenergetics and Is Protective in Parkinson's Disease

Journal: Antioxidants & Redox Signaling

doi: 10.1089/ars.2017.6992

Nitrite administration ameliorates pathology in rotenone and 6-OHDA rat models of PD . (A) Schematic of the experimental paradigm for nitrite and rotenone administration. (B) Representative image of striatal TH immunohistochemistry reveals severe depletion in DA fibers after rotenone exposure, which is strongly mitigated on treatment with inorganic nitrite (scale bar: 1 mm). (C) Representative image of rotenone-induced DA loss in the SNpc, which is prevented by nitrite co-treatment (scale bar: 100 μm). (D) Quantification of striatal innervation in (B) by densitometry. (E) Quantification of DA loss in the SNpc (C) by unbiased stereological counts. (F) Experimental 6-OHDA setup: In the acute paradigm ( top ), nitrite was administered 24 and 1 h before stereotactic infusion in the rat striatum; in the chronic paradigm ( bottom ), nitrite was administered orally, dissolved in drinking water, starting 7 days after induction of the lesion. (G, H) Nitrite administration ameliorates the 6-OHDA-induced lesions in striata and in SNpc. (I) Representative images of striata cross-sections injected with 6-OHDA ( ipsilateral ) and the respective untreated side ( contralateral ) of nitrite-treated rats (Scale bar: 1 mm) (* p
Figure Legend Snippet: Nitrite administration ameliorates pathology in rotenone and 6-OHDA rat models of PD . (A) Schematic of the experimental paradigm for nitrite and rotenone administration. (B) Representative image of striatal TH immunohistochemistry reveals severe depletion in DA fibers after rotenone exposure, which is strongly mitigated on treatment with inorganic nitrite (scale bar: 1 mm). (C) Representative image of rotenone-induced DA loss in the SNpc, which is prevented by nitrite co-treatment (scale bar: 100 μm). (D) Quantification of striatal innervation in (B) by densitometry. (E) Quantification of DA loss in the SNpc (C) by unbiased stereological counts. (F) Experimental 6-OHDA setup: In the acute paradigm ( top ), nitrite was administered 24 and 1 h before stereotactic infusion in the rat striatum; in the chronic paradigm ( bottom ), nitrite was administered orally, dissolved in drinking water, starting 7 days after induction of the lesion. (G, H) Nitrite administration ameliorates the 6-OHDA-induced lesions in striata and in SNpc. (I) Representative images of striata cross-sections injected with 6-OHDA ( ipsilateral ) and the respective untreated side ( contralateral ) of nitrite-treated rats (Scale bar: 1 mm) (* p

Techniques Used: Immunohistochemistry, Injection

22) Product Images from "Synergy Between Glutathione Peroxidase-1 and Astrocytic Growth Factors Suppresses Free Radical Generation and Protects Dopaminergic Neurons against 6-Hydroxydopamine"

Article Title: Synergy Between Glutathione Peroxidase-1 and Astrocytic Growth Factors Suppresses Free Radical Generation and Protects Dopaminergic Neurons against 6-Hydroxydopamine

Journal: Rejuvenation Research

doi: 10.1089/rej.2010.1080

Glutathione peroxidase-1 (GPX-1)–mediated protection of SK-N-MC cells against 6-hydroxydopamine (6-OHDA) toxicity. ( A ) Infected cells were treated with 200 μM 6-OHDA for 48 h and their images were collected. A fraction of pLV-GPX1–infected cells were also fed with astro-CM (CM). ( B ) 6-OHDA–treated cells in both the absence and presence of CM were tested for their viability using the MTT reduction assay. Each column represents the average of three independent experiments carried out in triplicate. Statistical differences in cell survival rate between untreated and CM-treated cells within the test group (pLV-GPX1 cells) are indicated by aa ( p
Figure Legend Snippet: Glutathione peroxidase-1 (GPX-1)–mediated protection of SK-N-MC cells against 6-hydroxydopamine (6-OHDA) toxicity. ( A ) Infected cells were treated with 200 μM 6-OHDA for 48 h and their images were collected. A fraction of pLV-GPX1–infected cells were also fed with astro-CM (CM). ( B ) 6-OHDA–treated cells in both the absence and presence of CM were tested for their viability using the MTT reduction assay. Each column represents the average of three independent experiments carried out in triplicate. Statistical differences in cell survival rate between untreated and CM-treated cells within the test group (pLV-GPX1 cells) are indicated by aa ( p

Techniques Used: Infection, MTT Assay

23) Product Images from "Characterization of Induced Pluripotent Stem Cell-derived Human Serotonergic Neurons"

Article Title: Characterization of Induced Pluripotent Stem Cell-derived Human Serotonergic Neurons

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2017.00131

Human induced pluripotent stem cells (iPSCs)-derived serotonergic neurons are sensitive to 5,7-DHT in vitro . (A) Phase contrast images and immunostaining images of the cells treated with different concentrations of 5,7-DHT. (B) Quantification of Serotonin positive or TH positive cells in (A) ( n = 3). Data are represented as mean ± SEM. (C) Phase contrast images and immunostaining images of the cells treated with different concentrations of 6-OHDA. (D) Quantification of Serotonin positive or TH positive cells in (C) ( n = 3). Data are represented as mean ± SEM. * P
Figure Legend Snippet: Human induced pluripotent stem cells (iPSCs)-derived serotonergic neurons are sensitive to 5,7-DHT in vitro . (A) Phase contrast images and immunostaining images of the cells treated with different concentrations of 5,7-DHT. (B) Quantification of Serotonin positive or TH positive cells in (A) ( n = 3). Data are represented as mean ± SEM. (C) Phase contrast images and immunostaining images of the cells treated with different concentrations of 6-OHDA. (D) Quantification of Serotonin positive or TH positive cells in (C) ( n = 3). Data are represented as mean ± SEM. * P

Techniques Used: Derivative Assay, In Vitro, Immunostaining

24) Product Images from "Integrated transcriptome expression profiling reveals a novel lncRNA associated with L-DOPA-induced dyskinesia in a rat model of Parkinson’s disease"

Article Title: Integrated transcriptome expression profiling reveals a novel lncRNA associated with L-DOPA-induced dyskinesia in a rat model of Parkinson’s disease

Journal: Aging (Albany NY)

doi: 10.18632/aging.102652

Validation of the rat model of PD and LID. ( A ) Experimental timeline showing 6-OHDA lesioning, L -DOPA administration, behavioral testing, and animal grouping. ( B ) Time course of the manifestation of AIMs scored every 3 days over a period of 21 days after the final L -DOPA administration (n = 15). ( C ) Representative photomicrographs of TH immunohistochemical staining in coronal brain sections of the striatum and SN of rats subjected to 6-OHDA injection into the right striatum (PD) with (LID) or without (NLID) L -DOPA administration. Magnified images correspond to labeled boxes in the upper panels (n = 3). ( D ) Quantification of TH expression in the striatum and SN and of c-FOS, p-ERK, and ERK expression in the striatum of PD and LID rats and their corresponding control groups (n = 3–5). The signal intensity of protein bands was normalized to that of GAPDH. ( E ) qRT-PCR detection of c-Fos expression in the striatum of PD and LID rats and their corresponding controls. Data are shown as mean ± SEM (n = 6). **P
Figure Legend Snippet: Validation of the rat model of PD and LID. ( A ) Experimental timeline showing 6-OHDA lesioning, L -DOPA administration, behavioral testing, and animal grouping. ( B ) Time course of the manifestation of AIMs scored every 3 days over a period of 21 days after the final L -DOPA administration (n = 15). ( C ) Representative photomicrographs of TH immunohistochemical staining in coronal brain sections of the striatum and SN of rats subjected to 6-OHDA injection into the right striatum (PD) with (LID) or without (NLID) L -DOPA administration. Magnified images correspond to labeled boxes in the upper panels (n = 3). ( D ) Quantification of TH expression in the striatum and SN and of c-FOS, p-ERK, and ERK expression in the striatum of PD and LID rats and their corresponding control groups (n = 3–5). The signal intensity of protein bands was normalized to that of GAPDH. ( E ) qRT-PCR detection of c-Fos expression in the striatum of PD and LID rats and their corresponding controls. Data are shown as mean ± SEM (n = 6). **P

Techniques Used: Immunohistochemistry, Staining, Injection, Labeling, Expressing, Quantitative RT-PCR

25) Product Images from "Secretome released from hydrogel-embedded adipose mesenchymal stem cells protects against the Parkinson’s disease related toxin 6-hydroxydopamine"

Article Title: Secretome released from hydrogel-embedded adipose mesenchymal stem cells protects against the Parkinson’s disease related toxin 6-hydroxydopamine

Journal: European Journal of Pharmaceutics and Biopharmaceutics

doi: 10.1016/j.ejpb.2017.09.014

Cytocompatibility and antioxidant effect of conditioned media (CM) from hydrogel-embedded RAA-MSCs. (A) Metabolic activity of SH-SY5Y cells after 24 h incubation with CM from RAA-MSCs cells included in hydrogels. Statistical analysis was done considering αMEM without FBS as control. One-way ANOVA followed by Tukey's multiple comparisons test (mean ± SD, n = 5). * p = 0.0226 and 0.025, and ns: not significant. (B) Metabolic activity of SH-SY5Y cells exposed to hydrogel-embedded RAA-MSC CM, collected after 24 h conditioning followed by 24 h challenge with 6-OHDA. Results are mean ± SD (n = 12). Statistical analysis was done using two-way ANOVA followed by Tukey's multiple comparisons test: * p = 0.012; **** p
Figure Legend Snippet: Cytocompatibility and antioxidant effect of conditioned media (CM) from hydrogel-embedded RAA-MSCs. (A) Metabolic activity of SH-SY5Y cells after 24 h incubation with CM from RAA-MSCs cells included in hydrogels. Statistical analysis was done considering αMEM without FBS as control. One-way ANOVA followed by Tukey's multiple comparisons test (mean ± SD, n = 5). * p = 0.0226 and 0.025, and ns: not significant. (B) Metabolic activity of SH-SY5Y cells exposed to hydrogel-embedded RAA-MSC CM, collected after 24 h conditioning followed by 24 h challenge with 6-OHDA. Results are mean ± SD (n = 12). Statistical analysis was done using two-way ANOVA followed by Tukey's multiple comparisons test: * p = 0.012; **** p

Techniques Used: Activity Assay, Incubation

Concentration-dependent effect and specificity of RAA-MSC conditioned media (CM) against oxidative stress. (A). Dose-response to 6-hydroxydopamine (6-OHDA) 100 μM of decreasing dilutions of RAA-MSCs CM. SH-SY5Y were pre-conditioned for 24 h before challenge with the toxin for a further 24 h. Cell viability was quantified by MTS assay. (B) The CM from dermal fibroblasts was unable to prevent the oxidative damage triggered by 6-OHDA. **** p
Figure Legend Snippet: Concentration-dependent effect and specificity of RAA-MSC conditioned media (CM) against oxidative stress. (A). Dose-response to 6-hydroxydopamine (6-OHDA) 100 μM of decreasing dilutions of RAA-MSCs CM. SH-SY5Y were pre-conditioned for 24 h before challenge with the toxin for a further 24 h. Cell viability was quantified by MTS assay. (B) The CM from dermal fibroblasts was unable to prevent the oxidative damage triggered by 6-OHDA. **** p

Techniques Used: Concentration Assay, MTS Assay

Contribution to neuroprotection of the proliferative effect of conditioned medium (CM) of hydrogel-embedded RAA-MSCs (A). Concentration of DNA in the SH-SY5Y cells treated with CM quantified by Hoechst. One-way ANOVA followed by Tukey's multiple comparisons test (mean ± SD, n = 6). ** p = 0.0011. (B) Metabolic activity of SH-SY5Y cells exposed to CM from hydrogel embedded RAA-MSCs collected after 48 h of conditioning. Cells were the exposed for 24 h to oxidative stress triggered by 6-OHDA 75 μM. The results are weighted on the relative cell proliferation compared to the control without FBS (mean ± SD, n = 5). One-way ANOVA followed by Tukey 's multiple comparisons test: **** p
Figure Legend Snippet: Contribution to neuroprotection of the proliferative effect of conditioned medium (CM) of hydrogel-embedded RAA-MSCs (A). Concentration of DNA in the SH-SY5Y cells treated with CM quantified by Hoechst. One-way ANOVA followed by Tukey's multiple comparisons test (mean ± SD, n = 6). ** p = 0.0011. (B) Metabolic activity of SH-SY5Y cells exposed to CM from hydrogel embedded RAA-MSCs collected after 48 h of conditioning. Cells were the exposed for 24 h to oxidative stress triggered by 6-OHDA 75 μM. The results are weighted on the relative cell proliferation compared to the control without FBS (mean ± SD, n = 5). One-way ANOVA followed by Tukey 's multiple comparisons test: **** p

Techniques Used: Concentration Assay, Activity Assay

26) Product Images from "Functional Reorganization of Motor and Limbic Circuits after Exercise Training in a Rat Model of Bilateral Parkinsonism"

Article Title: Functional Reorganization of Motor and Limbic Circuits after Exercise Training in a Rat Model of Bilateral Parkinsonism

Journal: PLoS ONE

doi: 10.1371/journal.pone.0080058

6-OHDA lesions. Shown are the effect of bilateral striatal lesions on (A) lesion volume (% of striatal volume), (B) tyrosine hydroxylase (TH) staining of the striatum, (C) TH staining of the substantia nigra reticulata (SNR), and (D) TH staining of the substantia nigra compacta (SNC). #: significant difference between sham rats without exercise training (Sham/No-ET) and lesioned rats without ET (Lesion/No-ET), P
Figure Legend Snippet: 6-OHDA lesions. Shown are the effect of bilateral striatal lesions on (A) lesion volume (% of striatal volume), (B) tyrosine hydroxylase (TH) staining of the striatum, (C) TH staining of the substantia nigra reticulata (SNR), and (D) TH staining of the substantia nigra compacta (SNC). #: significant difference between sham rats without exercise training (Sham/No-ET) and lesioned rats without ET (Lesion/No-ET), P

Techniques Used: Staining

27) Product Images from "LncRNA H19 diminishes dopaminergic neuron loss by mediating microRNA-301b-3p in Parkinson’s disease via the HPRT1-mediated Wnt/β-catenin signaling pathway"

Article Title: LncRNA H19 diminishes dopaminergic neuron loss by mediating microRNA-301b-3p in Parkinson’s disease via the HPRT1-mediated Wnt/β-catenin signaling pathway

Journal: Aging (Albany NY)

doi: 10.18632/aging.102877

Overexpressed HPRT1 activates the Wnt/β-catenin signaling pathway in a PD model. 6-OHDA-treated N27 dopaminergic neurons were treated with oe-NC or oe-HPRT1. ( A ) The protein expression of total-β-catenin and DAT as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio using TOP/FOP flash reporter assay. FOP was designated as background value or as negative control due to its stability. * p
Figure Legend Snippet: Overexpressed HPRT1 activates the Wnt/β-catenin signaling pathway in a PD model. 6-OHDA-treated N27 dopaminergic neurons were treated with oe-NC or oe-HPRT1. ( A ) The protein expression of total-β-catenin and DAT as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio using TOP/FOP flash reporter assay. FOP was designated as background value or as negative control due to its stability. * p

Techniques Used: Expressing, Western Blot, Activity Assay, Reporter Assay, Negative Control

Schematic representation of H19 in regulating dopaminergic neuron loss in PD. H19 upregulates the expression of HPRT1 by binding to miR-301b-3p. Overexpression of HPRT1 could activate the Wnt/β-catenin signaling pathway, thus promoting the mRNA expression of Nurr-1, Pitx-3, Ngn-2 and NeuroD1 in the substantia nigra tissues, which ultimately rescues the dopaminergic neuron loss in this 6-OHDA-induced PD mouse model.
Figure Legend Snippet: Schematic representation of H19 in regulating dopaminergic neuron loss in PD. H19 upregulates the expression of HPRT1 by binding to miR-301b-3p. Overexpression of HPRT1 could activate the Wnt/β-catenin signaling pathway, thus promoting the mRNA expression of Nurr-1, Pitx-3, Ngn-2 and NeuroD1 in the substantia nigra tissues, which ultimately rescues the dopaminergic neuron loss in this 6-OHDA-induced PD mouse model.

Techniques Used: Expressing, Binding Assay, Over Expression

Overexpressed HPRT1 inhibits dopaminergic neuron loss in 6-OHDA-induced PD mice. 6-OHDA-induced PD mice were treated with oe-NC or oe-HPRT1. ( A ) Protein expression of HPRT1 in the substantia nigra tissues examined by western blot assay. ( B ) TH positive neurons in the substantia nigra examined by immunohistochemistry (upper × 100, lower × 400). ( C ) Fluoro-Jade B-stained apoptotic neurons (scale bar = 50 μm). ( D – G ) The mRNA expression of Nurr-1 ( D ), Pitx-3 ( E ), Ngn-2 ( F ) and NeuroD1 ( G ) in the substantia nigra tissues examined by RT-qPCR. * p
Figure Legend Snippet: Overexpressed HPRT1 inhibits dopaminergic neuron loss in 6-OHDA-induced PD mice. 6-OHDA-induced PD mice were treated with oe-NC or oe-HPRT1. ( A ) Protein expression of HPRT1 in the substantia nigra tissues examined by western blot assay. ( B ) TH positive neurons in the substantia nigra examined by immunohistochemistry (upper × 100, lower × 400). ( C ) Fluoro-Jade B-stained apoptotic neurons (scale bar = 50 μm). ( D – G ) The mRNA expression of Nurr-1 ( D ), Pitx-3 ( E ), Ngn-2 ( F ) and NeuroD1 ( G ) in the substantia nigra tissues examined by RT-qPCR. * p

Techniques Used: Mouse Assay, Expressing, Western Blot, Immunohistochemistry, Staining, Quantitative RT-PCR

Overexpressed HPRT1 inhibits dopaminergic neuron loss via the Wnt/β-catenin signaling pathway in 6-OHDA-treated N27 dopaminergic neurons and mice. N27 dopaminergic neurons and mice were treated with oe-HPRT1 in the presence of the Wnt/β-catenin blocker XAV-939 or DMSO carrier. ( A ) The protein expression of total-β-catenin, DAT and HPRT1 as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio in the N27 dopaminergic neurons. FOP was designated as background value or as negative control due to its stability. ( C ) The protein expression of total-β-catenin, DAT and HPRT1 as well as the extent of β-catenin phosphorylation in the normal and 6-OHDA-lesioned PD mice. ( D ) TH positive neurons in the substantia nigra tissues examined by immunohistochemistry (upper × 100, lower × 400). ( E – H ) The mRNA expression of Nurr-1 ( E ), Pitx-3 ( F ), Ngn-2 ( G ) and NeuroD1 ( H ) in the substantia nigra tissues examined by RT-qPCR. ( I ) Fluoro-Jade B-stained apoptotic N27 dopaminergic neurons (scale bar = 50 μm). * p
Figure Legend Snippet: Overexpressed HPRT1 inhibits dopaminergic neuron loss via the Wnt/β-catenin signaling pathway in 6-OHDA-treated N27 dopaminergic neurons and mice. N27 dopaminergic neurons and mice were treated with oe-HPRT1 in the presence of the Wnt/β-catenin blocker XAV-939 or DMSO carrier. ( A ) The protein expression of total-β-catenin, DAT and HPRT1 as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio in the N27 dopaminergic neurons. FOP was designated as background value or as negative control due to its stability. ( C ) The protein expression of total-β-catenin, DAT and HPRT1 as well as the extent of β-catenin phosphorylation in the normal and 6-OHDA-lesioned PD mice. ( D ) TH positive neurons in the substantia nigra tissues examined by immunohistochemistry (upper × 100, lower × 400). ( E – H ) The mRNA expression of Nurr-1 ( E ), Pitx-3 ( F ), Ngn-2 ( G ) and NeuroD1 ( H ) in the substantia nigra tissues examined by RT-qPCR. ( I ) Fluoro-Jade B-stained apoptotic N27 dopaminergic neurons (scale bar = 50 μm). * p

Techniques Used: Mouse Assay, Expressing, Western Blot, Activity Assay, Negative Control, Immunohistochemistry, Quantitative RT-PCR, Staining

Overexpressed H19 activates the Wnt/β-catenin signaling pathway by inhibiting miR-301b-3p. N27 dopaminergic neurons exposed to 6-OHDA were treated with agomir-miR-301b-3p alone or in the presence of oe-H19. ( A ) The protein expression of total-β-catenin and HPRT1 as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio in the N27 dopaminergic neurons. *, p
Figure Legend Snippet: Overexpressed H19 activates the Wnt/β-catenin signaling pathway by inhibiting miR-301b-3p. N27 dopaminergic neurons exposed to 6-OHDA were treated with agomir-miR-301b-3p alone or in the presence of oe-H19. ( A ) The protein expression of total-β-catenin and HPRT1 as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio in the N27 dopaminergic neurons. *, p

Techniques Used: Expressing, Western Blot, Activity Assay

HPRT1 is poorly expressed in the substantia nigra tissues of 6-OHDA-induced PD mice. ( A ) The expression of HPRT1 in the expression profile of GSE20141 related to PD; ( B ) The expression of HPRT1 in the expression profile of GSE20168 related to PD. ( C ) The protein expression of TH in the substantia nigra tissues of 6-OHDA-induced PD mice measured by western blot analysis. ( D ) Immunohistochemical analysis for the TH positive cells in the substantia nigra tissues of 6-OHDA-induced PD mice (upper × 100, lower × 400); ( E ) Fluoro-Jade B-stained apoptotic neurons (scale bar = 50 μm). ( F ) mRNA expression of HPRT1 in the substantia nigra tissues examined by RT-qPCR; ( G ) Protein expression of HPRT1 in the substantia nigra tissues examined by western blot assay. * p
Figure Legend Snippet: HPRT1 is poorly expressed in the substantia nigra tissues of 6-OHDA-induced PD mice. ( A ) The expression of HPRT1 in the expression profile of GSE20141 related to PD; ( B ) The expression of HPRT1 in the expression profile of GSE20168 related to PD. ( C ) The protein expression of TH in the substantia nigra tissues of 6-OHDA-induced PD mice measured by western blot analysis. ( D ) Immunohistochemical analysis for the TH positive cells in the substantia nigra tissues of 6-OHDA-induced PD mice (upper × 100, lower × 400); ( E ) Fluoro-Jade B-stained apoptotic neurons (scale bar = 50 μm). ( F ) mRNA expression of HPRT1 in the substantia nigra tissues examined by RT-qPCR; ( G ) Protein expression of HPRT1 in the substantia nigra tissues examined by western blot assay. * p

Techniques Used: Mouse Assay, Expressing, Western Blot, Immunohistochemistry, Staining, Quantitative RT-PCR

H19 inhibits dopaminergic neuron loss by inhibiting miR-301b-3p. 6-OHDA-induced PD model mice were treated with agomir-miR-301b-3p alone or in the presence of oe-H19. ( A ) TH positive neurons in the substantia nigra tissues examined by immunohistochemistry (upper × 100, lower × 400). ( B – E ) The mRNA expression of Nurr-1 ( B ), Pitx-3 ( C ), Ngn-2 ( D ) and NeuroD1 ( E ) in the substantia nigra tissues examined by RT-qPCR. ( F ) Fluoro-Jade B-stained apoptotic neurons in the substantia nigra tissues (scale bar = 50 μm). * p
Figure Legend Snippet: H19 inhibits dopaminergic neuron loss by inhibiting miR-301b-3p. 6-OHDA-induced PD model mice were treated with agomir-miR-301b-3p alone or in the presence of oe-H19. ( A ) TH positive neurons in the substantia nigra tissues examined by immunohistochemistry (upper × 100, lower × 400). ( B – E ) The mRNA expression of Nurr-1 ( B ), Pitx-3 ( C ), Ngn-2 ( D ) and NeuroD1 ( E ) in the substantia nigra tissues examined by RT-qPCR. ( F ) Fluoro-Jade B-stained apoptotic neurons in the substantia nigra tissues (scale bar = 50 μm). * p

Techniques Used: Mouse Assay, Immunohistochemistry, Expressing, Quantitative RT-PCR, Staining

28) Product Images from "LRRK2 phosphorylation level correlates with abnormal motor behaviour in an experimental model of levodopa-induced dyskinesias"

Article Title: LRRK2 phosphorylation level correlates with abnormal motor behaviour in an experimental model of levodopa-induced dyskinesias

Journal: Molecular Brain

doi: 10.1186/s13041-016-0234-2

Effects of LRRK2 inhibitors on dyskinesia in 6-OHDA parkinsonian rats. a The panel is a schematic representation of the experimental design. Rats were unilaterally injected with 6-OHDA in the left MFB. After 15 days, the extent of the lesion was evaluated upon apomorphine answer. Two months after the 6-OHDA injection, fully lesioned rats ( > 200 controlateral turns) were chronically treated with L-DOPA. LRRK2 inhibitors were administrated to low dyskinetic animals with a single intrastriatal (i.s.) injection after 2 weeks of L-DOPA treatment. LIDs onset and severity were evaluated by AIMs scoring. b IN-1 (5 nmol), LRRK2 inhibitor III (5 nmol) or vehicle (5 μl 0.5 % DMSO) were stereotaxically injected in the ipsilateral striatum of low dyskinetic rats. I.s. injections were performed 6 h before the daily L-DOPA administration and the evaluation of AIMs were carried out from 20 to 140 min after L-DOPA. Both inhibitors were able to induce a significant increase in the AIMs score compared to vehicle injected rats and to pre-surgery values ( n = 7; −18 h: *** p
Figure Legend Snippet: Effects of LRRK2 inhibitors on dyskinesia in 6-OHDA parkinsonian rats. a The panel is a schematic representation of the experimental design. Rats were unilaterally injected with 6-OHDA in the left MFB. After 15 days, the extent of the lesion was evaluated upon apomorphine answer. Two months after the 6-OHDA injection, fully lesioned rats ( > 200 controlateral turns) were chronically treated with L-DOPA. LRRK2 inhibitors were administrated to low dyskinetic animals with a single intrastriatal (i.s.) injection after 2 weeks of L-DOPA treatment. LIDs onset and severity were evaluated by AIMs scoring. b IN-1 (5 nmol), LRRK2 inhibitor III (5 nmol) or vehicle (5 μl 0.5 % DMSO) were stereotaxically injected in the ipsilateral striatum of low dyskinetic rats. I.s. injections were performed 6 h before the daily L-DOPA administration and the evaluation of AIMs were carried out from 20 to 140 min after L-DOPA. Both inhibitors were able to induce a significant increase in the AIMs score compared to vehicle injected rats and to pre-surgery values ( n = 7; −18 h: *** p

Techniques Used: Injection

29) Product Images from "Role of nucleus of the solitary tract noradrenergic neurons in post-stress cardiovascular and hormonal control in male rats"

Article Title: Role of nucleus of the solitary tract noradrenergic neurons in post-stress cardiovascular and hormonal control in male rats

Journal: Stress (Amsterdam, Netherlands)

doi: 10.3109/10253890.2015.1013531

Cardiovascular responses to novel stress in chronically stressed vehicle- and 6-OHDA-treated rats
Figure Legend Snippet: Cardiovascular responses to novel stress in chronically stressed vehicle- and 6-OHDA-treated rats

Techniques Used:

Body weight, adrenal weight, and thymus weight following chronic variable stress (CVS) in 6-OHDA and vehicle rats
Figure Legend Snippet: Body weight, adrenal weight, and thymus weight following chronic variable stress (CVS) in 6-OHDA and vehicle rats

Techniques Used:

Time course of changes in heart rate variability (HRV) parameters during chronic variable stress in vehicle- and 6-OHDA-treated rats
Figure Legend Snippet: Time course of changes in heart rate variability (HRV) parameters during chronic variable stress in vehicle- and 6-OHDA-treated rats

Techniques Used:

30) Product Images from "Intrastriatal Transplantation of Adult Human Neural Crest-Derived Stem Cells Improves Functional Outcome in Parkinsonian Rats"

Article Title: Intrastriatal Transplantation of Adult Human Neural Crest-Derived Stem Cells Improves Functional Outcome in Parkinsonian Rats

Journal: Stem Cells Translational Medicine

doi: 10.5966/sctm.2014-0078

Transplantation of ITSCs into 6-hydroxydopamine (6-OHDA)-lesioned parkinsonian rats led to behavioral recovery. Relative amphetamine-induced rotational behavior of 6-OHDA lesioned rats at baseline (before engraftment) and at 4, 8, and 12 weeks after intrastriatal transplantation of ITSCs cultured under standard conditions ( n = 7) or pretreated with SHH/FGF8 ( n = 8) and in sham-operated animals ( n = 5). Baseline values were 12.9 ± 1.5 net rotations per minute for the sham animal group, 13.7 ± 0.5 for the standard-ITSC group, and 15.9 ± 3.8 for the pretreated-ITSC group (F = 2.029; p = .156, one-way analysis of variance [ANOVA]). Two-way repeated-measures ANOVA with post hoc t test and Bonferroni adjustment with time after transplantation as within-subject factor and treatment as between-subjects factor revealed a statistically significant main effect for time (F[2.155, 38.781] = 10.947, p
Figure Legend Snippet: Transplantation of ITSCs into 6-hydroxydopamine (6-OHDA)-lesioned parkinsonian rats led to behavioral recovery. Relative amphetamine-induced rotational behavior of 6-OHDA lesioned rats at baseline (before engraftment) and at 4, 8, and 12 weeks after intrastriatal transplantation of ITSCs cultured under standard conditions ( n = 7) or pretreated with SHH/FGF8 ( n = 8) and in sham-operated animals ( n = 5). Baseline values were 12.9 ± 1.5 net rotations per minute for the sham animal group, 13.7 ± 0.5 for the standard-ITSC group, and 15.9 ± 3.8 for the pretreated-ITSC group (F = 2.029; p = .156, one-way analysis of variance [ANOVA]). Two-way repeated-measures ANOVA with post hoc t test and Bonferroni adjustment with time after transplantation as within-subject factor and treatment as between-subjects factor revealed a statistically significant main effect for time (F[2.155, 38.781] = 10.947, p

Techniques Used: Transplantation Assay, Cell Culture

Lesioned substantia nigra (SN) of rats treated with 6-hydroxydopamine (6-OHDA) is restored after ITSC transplantation. (A): Contralateral hemispheres revealed normal endogenous populations of TH-positive DA neurons in the SN. Although almost no TH-expressing DA neurons were observable after transplantation of the sham group, populations of DA neurons were observable after transplantation of undifferentiated or predifferentiated ITSCs. (B): TH expression within the striatum contralateral and ipsilateral to an unilateral striatal 6-OHDA lesion 12 weeks after lesion. The 6-OHDA lesion affected TH + fibers only within the ipsilateral hemisphere. In contrast to the sham group, in transplanted animals, TH + fibers are clearly visible within the ipsilateral hemisphere (magnified view). (C, D): Box plots of TH + DA neuron counts in the SN on ipsilateral (C) and contralateral (D) sides with respect to the transplantation group, as measured by stereological analysis. The plots show the 10th percentile, 1st quartile, median, 3rd quartile, and 90th percentile for each parameter. Open circles represent the means. The p values are from one-way ANOVA according to Kruskal-Wallis. (E): Significantly increased amount of incoming endogenous TH+ fibers into ipsilateral striatum after transplantation of undifferentiated and SHH/FGF8-pretreated ITSCs in contrast to sham control. The p values are calculated by one-way ANOVA with post hoc Bonferroni-adjusted t test. (F, G): Correlations of ipsilateral amphetamine-induced rotations after 12 weeks with ipsilateral (F) and contralateral (G) TH + DA neuron counts. Values are means ± SEM. Abbreviations: DA, dopaminergic; ITSCs, adult human neural crest-derived stem cells derived from the inferior turbinate; TH, tyrosine hydroxylase.
Figure Legend Snippet: Lesioned substantia nigra (SN) of rats treated with 6-hydroxydopamine (6-OHDA) is restored after ITSC transplantation. (A): Contralateral hemispheres revealed normal endogenous populations of TH-positive DA neurons in the SN. Although almost no TH-expressing DA neurons were observable after transplantation of the sham group, populations of DA neurons were observable after transplantation of undifferentiated or predifferentiated ITSCs. (B): TH expression within the striatum contralateral and ipsilateral to an unilateral striatal 6-OHDA lesion 12 weeks after lesion. The 6-OHDA lesion affected TH + fibers only within the ipsilateral hemisphere. In contrast to the sham group, in transplanted animals, TH + fibers are clearly visible within the ipsilateral hemisphere (magnified view). (C, D): Box plots of TH + DA neuron counts in the SN on ipsilateral (C) and contralateral (D) sides with respect to the transplantation group, as measured by stereological analysis. The plots show the 10th percentile, 1st quartile, median, 3rd quartile, and 90th percentile for each parameter. Open circles represent the means. The p values are from one-way ANOVA according to Kruskal-Wallis. (E): Significantly increased amount of incoming endogenous TH+ fibers into ipsilateral striatum after transplantation of undifferentiated and SHH/FGF8-pretreated ITSCs in contrast to sham control. The p values are calculated by one-way ANOVA with post hoc Bonferroni-adjusted t test. (F, G): Correlations of ipsilateral amphetamine-induced rotations after 12 weeks with ipsilateral (F) and contralateral (G) TH + DA neuron counts. Values are means ± SEM. Abbreviations: DA, dopaminergic; ITSCs, adult human neural crest-derived stem cells derived from the inferior turbinate; TH, tyrosine hydroxylase.

Techniques Used: Transplantation Assay, Expressing, Derivative Assay

Adult human neural crest-derived stem cells derived from the inferior turbinate (ITSCs) survive and integrate into parkinsonian rat brains. Immunohistochemical analysis of sagittal 6-hydroxydopamine (6-OHDA) rat brain sections at 12 weeks after transplantation. (A): Whole-brain section stained with DAPI (4′,6-diamidino-2-phenylindole). Dotted line indicates AP axis of injection site. Squares depicting regions of interest with appropriate migration distances. (B): Cluster of STEM121 + cells were directly located at the striatal injection site (upper panel). Dotted ellipse illustrates the injection channel. GFAP + astrocytes were recruited to the injury site. Magnified view: arrowhead indicates STEM121 + /GFAP + coexpressing cell (lower panel). (C): ITSCs remaining at the injection site kept their stem cell characteristics and expressed the human-specific neural crest stem cell marker nestin. (D): STEM121 + /Map2 + ITSCs were shown to be located within the corpus callosum directly above the striatum. (E): At 12 weeks after transplantation, several STEM121 + cells remained within the corpus callosum 1.6 mm posterior to the injection site. (F): In addition, STEM121 + ITSCs were located adjacent to the injection channel of the 6-OHDA lesion, indicated by dotted lines (G), and 7.3 mm posterior to the striatal injection site within the midbrain. (H): STEM121 + /Map2 + ITSCs integrated directly above the lesioned SN. (I): Stem121 + ITSCs located in the locus ceruleus showed TH coexpression at the protein level, indicating their differentiation into DA neurons. Abbreviations: AP, anteroposterior; hNestin, human-specific neural crest stem cell marker nestin; TH, tyrosine hydroxylase.
Figure Legend Snippet: Adult human neural crest-derived stem cells derived from the inferior turbinate (ITSCs) survive and integrate into parkinsonian rat brains. Immunohistochemical analysis of sagittal 6-hydroxydopamine (6-OHDA) rat brain sections at 12 weeks after transplantation. (A): Whole-brain section stained with DAPI (4′,6-diamidino-2-phenylindole). Dotted line indicates AP axis of injection site. Squares depicting regions of interest with appropriate migration distances. (B): Cluster of STEM121 + cells were directly located at the striatal injection site (upper panel). Dotted ellipse illustrates the injection channel. GFAP + astrocytes were recruited to the injury site. Magnified view: arrowhead indicates STEM121 + /GFAP + coexpressing cell (lower panel). (C): ITSCs remaining at the injection site kept their stem cell characteristics and expressed the human-specific neural crest stem cell marker nestin. (D): STEM121 + /Map2 + ITSCs were shown to be located within the corpus callosum directly above the striatum. (E): At 12 weeks after transplantation, several STEM121 + cells remained within the corpus callosum 1.6 mm posterior to the injection site. (F): In addition, STEM121 + ITSCs were located adjacent to the injection channel of the 6-OHDA lesion, indicated by dotted lines (G), and 7.3 mm posterior to the striatal injection site within the midbrain. (H): STEM121 + /Map2 + ITSCs integrated directly above the lesioned SN. (I): Stem121 + ITSCs located in the locus ceruleus showed TH coexpression at the protein level, indicating their differentiation into DA neurons. Abbreviations: AP, anteroposterior; hNestin, human-specific neural crest stem cell marker nestin; TH, tyrosine hydroxylase.

Techniques Used: Derivative Assay, Immunohistochemistry, Transplantation Assay, Staining, Injection, Migration, Marker

31) Product Images from "Region-Specific Neuroprotective Features of Astrocytes against Oxidative Stress Induced by 6-Hydroxydopamine"

Article Title: Region-Specific Neuroprotective Features of Astrocytes against Oxidative Stress Induced by 6-Hydroxydopamine

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20030598

( A ) Neuroprotective effects of co-culture with mesencephalic or striatal astrocytes against 6-hydroxydopamine (6-OHDA)-induced toxicity in mesencephalic neurons. Mesencephalic neurons were exposed to 6-OHDA (50 µM) for 24 h co-existing with mesencephalic or striatal astrocytes. Each value is mean of the number of tyrosine hydroxylase (TH)-positive neurons ± SEM ( n = 4); ** p
Figure Legend Snippet: ( A ) Neuroprotective effects of co-culture with mesencephalic or striatal astrocytes against 6-hydroxydopamine (6-OHDA)-induced toxicity in mesencephalic neurons. Mesencephalic neurons were exposed to 6-OHDA (50 µM) for 24 h co-existing with mesencephalic or striatal astrocytes. Each value is mean of the number of tyrosine hydroxylase (TH)-positive neurons ± SEM ( n = 4); ** p

Techniques Used: Co-Culture Assay

( A ) Regional difference of glia conditioned media (GCM). Cell viability of TH-positive dopaminergic neurons co-incubated with mesencephalic or striatal GCM (control-GCM, 6-OHDA-GCM, GCM with 6-OHDA (100 µM) for 24 h. Each value is mean ± SEM ( n = 4) expressed as percentage of each control-GCM group; ** p
Figure Legend Snippet: ( A ) Regional difference of glia conditioned media (GCM). Cell viability of TH-positive dopaminergic neurons co-incubated with mesencephalic or striatal GCM (control-GCM, 6-OHDA-GCM, GCM with 6-OHDA (100 µM) for 24 h. Each value is mean ± SEM ( n = 4) expressed as percentage of each control-GCM group; ** p

Techniques Used: Incubation

( A , B ) Effects of 6-OHDA treatment (100–150 µM) for 6 h on nuclear Nrf2 expression in mesencephalic ( A ) or striatal ( B ) astrocytes. Data are means ± SEM ( n = 5–6); * p
Figure Legend Snippet: ( A , B ) Effects of 6-OHDA treatment (100–150 µM) for 6 h on nuclear Nrf2 expression in mesencephalic ( A ) or striatal ( B ) astrocytes. Data are means ± SEM ( n = 5–6); * p

Techniques Used: Expressing

( A ) Representative fluorescence photomicrographs of dihydroethidium (DHE)-derived fluorescence (red) on cultured mesencephalic or striatal astrocytes treated with 6-OHDA (50–150 µM) for 24 h. Images are taken at 100× magnification. ( B ) Effect of 6-OHDA treatment on superoxide production in astrocytes. Signal intensity of DHE-positive astrocytes (mesencephalic or striatal astrocytes) treated with 6-OHDA (50–150 µM) for 24 h. Data are means ± SEM ( n = 6); * p
Figure Legend Snippet: ( A ) Representative fluorescence photomicrographs of dihydroethidium (DHE)-derived fluorescence (red) on cultured mesencephalic or striatal astrocytes treated with 6-OHDA (50–150 µM) for 24 h. Images are taken at 100× magnification. ( B ) Effect of 6-OHDA treatment on superoxide production in astrocytes. Signal intensity of DHE-positive astrocytes (mesencephalic or striatal astrocytes) treated with 6-OHDA (50–150 µM) for 24 h. Data are means ± SEM ( n = 6); * p

Techniques Used: Fluorescence, Derivative Assay, Cell Culture

( A ) Changes in quinoprotein formation in mesencephalic neurons co-cultured with mesencephalic or striatal astrocytes treated with 6-OHDA (100 µM) for 24 h. Each value is mean ± SEM ( n = 4); * p
Figure Legend Snippet: ( A ) Changes in quinoprotein formation in mesencephalic neurons co-cultured with mesencephalic or striatal astrocytes treated with 6-OHDA (100 µM) for 24 h. Each value is mean ± SEM ( n = 4); * p

Techniques Used: Cell Culture

Alterations of mRNA gene expression in mesencephalic or striatal astrocytes treated with 6-OHDA. Cultured mesencephalic or striatal astrocytes were treated with 6-OHDA (100 µM) for 24 h. The mRNA gene expression ratio was expressed in fluorescence intensity of 6-OHDA-treated group/fluorescence intensity of control group on a cDNA microarray. The increasing gene was defined as Log 2 (ratio) > 1 if ratio was more than double, and the decreasing gene was defined as Log 2 (ratio)
Figure Legend Snippet: Alterations of mRNA gene expression in mesencephalic or striatal astrocytes treated with 6-OHDA. Cultured mesencephalic or striatal astrocytes were treated with 6-OHDA (100 µM) for 24 h. The mRNA gene expression ratio was expressed in fluorescence intensity of 6-OHDA-treated group/fluorescence intensity of control group on a cDNA microarray. The increasing gene was defined as Log 2 (ratio) > 1 if ratio was more than double, and the decreasing gene was defined as Log 2 (ratio)

Techniques Used: Expressing, Cell Culture, Fluorescence, Microarray

Total glutathione levels in astrocytes treated with 6-OHDA. Mesencephalic ( A ) or striatal ( B ) astrocytes were treated with 6-OHDA (50–150 µM) for 24 h. Each value is presented as mean ± SEM ( n = 5–6); * p
Figure Legend Snippet: Total glutathione levels in astrocytes treated with 6-OHDA. Mesencephalic ( A ) or striatal ( B ) astrocytes were treated with 6-OHDA (50–150 µM) for 24 h. Each value is presented as mean ± SEM ( n = 5–6); * p

Techniques Used:

32) Product Images from "Bee Venom Alleviates Motor Deficits and Modulates the Transfer of Cortical Information through the Basal Ganglia in Rat Models of Parkinson’s Disease"

Article Title: Bee Venom Alleviates Motor Deficits and Modulates the Transfer of Cortical Information through the Basal Ganglia in Rat Models of Parkinson’s Disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0142838

Effect of systemic injection of bee venom on the pattern of responses evoked by cortical stimulation in a SNr neuron from a 6-OHDA lesioned rat. ( A ) Triphasic excitatory-inhibitory-excitatory sequence evoked in a SNr neuron by stimulation of the orofacial sensori-motor cortex in the 6-OHDA condition. ( B ) 30 minutes after systemic injection of bee venom (3 μg/kg), the inhibitory component of the response of the same SNr neuron was markedly increased, whereas the early and late excitatory components were not modified. Red numbers in A - C are as in Fig 4 . The same number of cortical stimulations (red bar, n = 70) was applied in A and B .
Figure Legend Snippet: Effect of systemic injection of bee venom on the pattern of responses evoked by cortical stimulation in a SNr neuron from a 6-OHDA lesioned rat. ( A ) Triphasic excitatory-inhibitory-excitatory sequence evoked in a SNr neuron by stimulation of the orofacial sensori-motor cortex in the 6-OHDA condition. ( B ) 30 minutes after systemic injection of bee venom (3 μg/kg), the inhibitory component of the response of the same SNr neuron was markedly increased, whereas the early and late excitatory components were not modified. Red numbers in A - C are as in Fig 4 . The same number of cortical stimulations (red bar, n = 70) was applied in A and B .

Techniques Used: Injection, Sequencing, Modification

Time schedule for bee venom injections and behavioral testing in 6-OHDA lesioned rats. Time points at which 6-OHDA lesion and bee venom (1 μg/kg for BV1 group or 3 μg/kg for BV3 group) or saline i.p. injections were carried out. Note that bee venom or saline was given every 3 days, starting 15–21 days after the 6-OHDA injection. Cyl. and Apo. indicate time points at which rats were taken for the cylinder test (Cyl.) or apomorphine-induced rotations (Apo.), respectively. After the apomorphine-induced rotations test, all rats were killed for brain processing and analysis.
Figure Legend Snippet: Time schedule for bee venom injections and behavioral testing in 6-OHDA lesioned rats. Time points at which 6-OHDA lesion and bee venom (1 μg/kg for BV1 group or 3 μg/kg for BV3 group) or saline i.p. injections were carried out. Note that bee venom or saline was given every 3 days, starting 15–21 days after the 6-OHDA injection. Cyl. and Apo. indicate time points at which rats were taken for the cylinder test (Cyl.) or apomorphine-induced rotations (Apo.), respectively. After the apomorphine-induced rotations test, all rats were killed for brain processing and analysis.

Techniques Used: Injection

33) Product Images from "Strain Difference in the Up-Regulation of FGF-2 Protein Following a Neurotoxic Lesion of the Nigrostriatal Pathway"

Article Title: Strain Difference in the Up-Regulation of FGF-2 Protein Following a Neurotoxic Lesion of the Nigrostriatal Pathway

Journal: Neurochemical research

doi: 10.1007/s11064-009-0093-7

Loss of dopamine in the ventral midbrain or striatum 1 week following a unilateral 6-OHDA lesion of the left nigrostriatal pathway. The amount of dopamine in each sample was determined by HPLC analysis. For each brain structure (ventral midbrain or striatum),
Figure Legend Snippet: Loss of dopamine in the ventral midbrain or striatum 1 week following a unilateral 6-OHDA lesion of the left nigrostriatal pathway. The amount of dopamine in each sample was determined by HPLC analysis. For each brain structure (ventral midbrain or striatum),

Techniques Used: High Performance Liquid Chromatography

Loss of dopamine in the ventral midbrain or striatum 3 weeks following a unilateral 6-OHDA lesion of the left nigrostriatal pathway. The amount of dopamine in each sample was determined by HPLC analysis. For each brain structure (ventral midbrain or striatum),
Figure Legend Snippet: Loss of dopamine in the ventral midbrain or striatum 3 weeks following a unilateral 6-OHDA lesion of the left nigrostriatal pathway. The amount of dopamine in each sample was determined by HPLC analysis. For each brain structure (ventral midbrain or striatum),

Techniques Used: High Performance Liquid Chromatography

34) Product Images from "Necdin and TrkA Contribute to Modulation by p75NTR of Resistance to Oxidant Stress"

Article Title: Necdin and TrkA Contribute to Modulation by p75NTR of Resistance to Oxidant Stress

Journal: Experimental cell research

doi: 10.1016/j.yexcr.2009.10.001

The effect of necdin siRNA on cell membrane integrity following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Trypan blue exclusion determinations were performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to those obtained in the absence of 6-OHDA for all conditions. Both p75NTR-native and p75NTR-deficient cell lines were treated with necdin or a scrambled control siRNA along with 6-OHDA treatment. *P
Figure Legend Snippet: The effect of necdin siRNA on cell membrane integrity following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Trypan blue exclusion determinations were performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to those obtained in the absence of 6-OHDA for all conditions. Both p75NTR-native and p75NTR-deficient cell lines were treated with necdin or a scrambled control siRNA along with 6-OHDA treatment. *P

Techniques Used: Concentration Assay

The effect of necdin siRNA and a JNK phosphorylation inhibitor, SP600125, on cell metabolic viability following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Alamar blue staining was performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. p75NTR-native and p75NTR-deficient cell lines were treated with necdin siRNA or a scrambled control siRNA with or without SP600125 and 6-OHDA treatment. *P values
Figure Legend Snippet: The effect of necdin siRNA and a JNK phosphorylation inhibitor, SP600125, on cell metabolic viability following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Alamar blue staining was performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. p75NTR-native and p75NTR-deficient cell lines were treated with necdin siRNA or a scrambled control siRNA with or without SP600125 and 6-OHDA treatment. *P values

Techniques Used: Staining, Concentration Assay

The effect of necdin siRNA on metabolic viability following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Alamar blue staining was performed as described in Materials and Methods and OD 590 nm values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. Both p75NTR-native and p75NTR-deficient cell lines were treated with necdin siRNA or a scrambled control siRNA along with 6-OHDA. *P
Figure Legend Snippet: The effect of necdin siRNA on metabolic viability following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells Alamar blue staining was performed as described in Materials and Methods and OD 590 nm values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. Both p75NTR-native and p75NTR-deficient cell lines were treated with necdin siRNA or a scrambled control siRNA along with 6-OHDA. *P

Techniques Used: Staining, Concentration Assay

The effect of necdin and p75NTR siRNAs on metabolic viability following 6-OHDA treatment of p75NTR-native PC12 cells Alamar blue staining was performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. p75NTR-native cells were treated with necdin siRNA, p75NTR siRNA, or a scrambled control siRNA along with 6-OHDA treatment. *P
Figure Legend Snippet: The effect of necdin and p75NTR siRNAs on metabolic viability following 6-OHDA treatment of p75NTR-native PC12 cells Alamar blue staining was performed as described in Materials and Methods and values obtained at each concentration of 6-OHDA were normalized to the value obtained in the absence of 6-OHDA for all conditions. p75NTR-native cells were treated with necdin siRNA, p75NTR siRNA, or a scrambled control siRNA along with 6-OHDA treatment. *P

Techniques Used: Staining, Concentration Assay

35) Product Images from "Intracellular delivery of Parkin rescues neurons from accumulation of damaged mitochondria and pathological α-synuclein"

Article Title: Intracellular delivery of Parkin rescues neurons from accumulation of damaged mitochondria and pathological α-synuclein

Journal: Science Advances

doi: 10.1126/sciadv.aba1193

Purification and characterization of iCP-Parkin; aMTD/SD-fused Parkin recombinant protein is cell permeable via direct cell-to-cell transfer. ( A ) Structure of Parkin recombinant proteins fused to aMTD524 and SDB (iCP-Parkin). SDS-PAGE (reducing and nonreducing) image of iCP-Parkin. ( B ) Neuronal (SH-SY5Y cell) measured by flow cytometry and confocal laser scanning microscopy, demonstrating the intracellular localization of iCP-Parkin. ( C ) Immunofluorescence staining data (green fluorescence) showing intracellular location of iCP-Parkin in primary neurons when cotreated with or without 6-OHDA at 2 and 24 hours. β-III-Tubulin was also costained and detected as red fluorescent signal. The representative data are presented, and the tests were carried out in quadruplicate ( n = 4). aMTD-mediated delivery of iCP-Parkin is notably affected by EDTA treatment ( D ) and low temperature ( E ) but unaffected by pretreatment of cells with the ATP-depleting agent antimycin ( F ), proteinase K ( G ), the microtubule inhibitor Taxol ( H ), a clathrin-mediated endocytosis blocker, chlorpromazine ( I ), a macropinocytosis blocker, amiloride ( J ), or a lipid raft–mediated endocytosis blocker, methyl-β-cyclodextrin ( K ). ( L ) Cell-to-cell transfer of iCP-Parkin. C2C12 cells (donor cells) were pretreated with FITC–iCP-Parkin (green) for 2 hours and were mixed with RAW264.7 cells (recipient cells) labeled with PE-CD14 antibody (red) for 2 hours. Green/red fluorescent double-positive cells were analyzed by flow cytometry. ( M ) The cytoprotective effect of iCP-Parkin via cell-to-cell transfer. iCP-Parkin–treated SH-SY5Y cells (for 2 hours) incubated with GFP-transfected SH-SY5Y cells for 6 hours. These mixed cells were treated with 2 mM MPP + for 24 hours. Apoptosis of GFP-positive cells was analyzed by an annexin V/7-AAD apoptosis detection assay. Quantification of cytoprotective effect by cell-to-cell transferred iCP-Parkin. Data are represented as the means ± SD with Student’s t test.
Figure Legend Snippet: Purification and characterization of iCP-Parkin; aMTD/SD-fused Parkin recombinant protein is cell permeable via direct cell-to-cell transfer. ( A ) Structure of Parkin recombinant proteins fused to aMTD524 and SDB (iCP-Parkin). SDS-PAGE (reducing and nonreducing) image of iCP-Parkin. ( B ) Neuronal (SH-SY5Y cell) measured by flow cytometry and confocal laser scanning microscopy, demonstrating the intracellular localization of iCP-Parkin. ( C ) Immunofluorescence staining data (green fluorescence) showing intracellular location of iCP-Parkin in primary neurons when cotreated with or without 6-OHDA at 2 and 24 hours. β-III-Tubulin was also costained and detected as red fluorescent signal. The representative data are presented, and the tests were carried out in quadruplicate ( n = 4). aMTD-mediated delivery of iCP-Parkin is notably affected by EDTA treatment ( D ) and low temperature ( E ) but unaffected by pretreatment of cells with the ATP-depleting agent antimycin ( F ), proteinase K ( G ), the microtubule inhibitor Taxol ( H ), a clathrin-mediated endocytosis blocker, chlorpromazine ( I ), a macropinocytosis blocker, amiloride ( J ), or a lipid raft–mediated endocytosis blocker, methyl-β-cyclodextrin ( K ). ( L ) Cell-to-cell transfer of iCP-Parkin. C2C12 cells (donor cells) were pretreated with FITC–iCP-Parkin (green) for 2 hours and were mixed with RAW264.7 cells (recipient cells) labeled with PE-CD14 antibody (red) for 2 hours. Green/red fluorescent double-positive cells were analyzed by flow cytometry. ( M ) The cytoprotective effect of iCP-Parkin via cell-to-cell transfer. iCP-Parkin–treated SH-SY5Y cells (for 2 hours) incubated with GFP-transfected SH-SY5Y cells for 6 hours. These mixed cells were treated with 2 mM MPP + for 24 hours. Apoptosis of GFP-positive cells was analyzed by an annexin V/7-AAD apoptosis detection assay. Quantification of cytoprotective effect by cell-to-cell transferred iCP-Parkin. Data are represented as the means ± SD with Student’s t test.

Techniques Used: Purification, Recombinant, SDS Page, Flow Cytometry, Confocal Laser Scanning Microscopy, Immunofluorescence, Staining, Fluorescence, Labeling, Incubation, Transfection, Detection Assay

iCP-Parkin ameliorates behavioral and molecular defects in 6-OHDA–induced PD animal models. ( A to F ) Efficacy of iCP-Parkin in a 6-OHDA–induced PD mouse model. (A) Schematic diagram of the experimental protocol. 6-OHDA (4 μg per head) was injected into the right side of the MFB. (B) Rotation test for confirming PD modeling at 3 days after 6-OHDA unilateral injection. (C) Pole test. After measuring the time to climb down a pole, raw data were converted into relative behavior activity based on the time of untreated control as 100%. * P
Figure Legend Snippet: iCP-Parkin ameliorates behavioral and molecular defects in 6-OHDA–induced PD animal models. ( A to F ) Efficacy of iCP-Parkin in a 6-OHDA–induced PD mouse model. (A) Schematic diagram of the experimental protocol. 6-OHDA (4 μg per head) was injected into the right side of the MFB. (B) Rotation test for confirming PD modeling at 3 days after 6-OHDA unilateral injection. (C) Pole test. After measuring the time to climb down a pole, raw data were converted into relative behavior activity based on the time of untreated control as 100%. * P

Techniques Used: Injection, Activity Assay

36) Product Images from "Blockade of fast A-type and TEA-sensitive potassium channels provide an antiparkinsonian effect in a 6-OHDA animal model"

Article Title: Blockade of fast A-type and TEA-sensitive potassium channels provide an antiparkinsonian effect in a 6-OHDA animal model

Journal: Neurosciences

doi: 10.17712/nsj.2017.1.20160266

- Time schedule used for our experiments: Animals were tested by apomorphine-induced rotational test and elevated body swing test (EBST) at fourth times: before stereotaxic surgery and 6-OHDA injection and in the third, fifth and eighth weeks after that. Rotational tests were performed at least 1 hour after termination of the EBST. Rotarod test, were performed in the seventh week after 6-OHDA injection. Blood sampling and measurement of its MDA concentration were performed before the surgery and in the fourth and ninth weeks after that. Pretreatments with potassium channel blockers of 4-AP and TEA and also saline was begun just before 6-OHDA injection and continued to seven days after that (black arrow). Numbers show the days after 6-OHDA injection. EBST - elevated body swing test, MDA - Malondialdehyde, 4-AP - 4-aminopyridine, TEA - Tetraethylammonium, 6-OHDA - 6-hydroxydopamine
Figure Legend Snippet: - Time schedule used for our experiments: Animals were tested by apomorphine-induced rotational test and elevated body swing test (EBST) at fourth times: before stereotaxic surgery and 6-OHDA injection and in the third, fifth and eighth weeks after that. Rotational tests were performed at least 1 hour after termination of the EBST. Rotarod test, were performed in the seventh week after 6-OHDA injection. Blood sampling and measurement of its MDA concentration were performed before the surgery and in the fourth and ninth weeks after that. Pretreatments with potassium channel blockers of 4-AP and TEA and also saline was begun just before 6-OHDA injection and continued to seven days after that (black arrow). Numbers show the days after 6-OHDA injection. EBST - elevated body swing test, MDA - Malondialdehyde, 4-AP - 4-aminopyridine, TEA - Tetraethylammonium, 6-OHDA - 6-hydroxydopamine

Techniques Used: Injection, Sampling, Multiple Displacement Amplification, Concentration Assay

37) Product Images from "Expression of nerve growth factor in the prostate of male rats in response to chronic stress and sympathetic denervation"

Article Title: Expression of nerve growth factor in the prostate of male rats in response to chronic stress and sympathetic denervation

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2014.1856

Expression of NGF in the prostatic lobes of male Wistar rats. (A) Ventral prostate of an untreated control rat. (B) Proliferation of the epithelium in the ventral prostate of a WIRS-treated rat, characterized by intraluminal villous enfolding, as well as the appearance of epithelial nodules, piling up of epithelial cells and a loss of cell polarity. NGF expression is increased. (C) Ventral prostate of a 6-OHDA-treated rat showing dilated acini and a significant increase in NGF level. (D) Ventral prostate of a WIRS plus 6-OHDA-treated rat without proliferation. (E) Negative control. All images were captured using the same magnification (original magnification, ×200; bar, 100 μm) and stained using Hematoxylin-Eosin. NGF, nerve growth factor; WIRS, restraint water-immersion stress; 6-OHDA, 6-hydroxydopamine.
Figure Legend Snippet: Expression of NGF in the prostatic lobes of male Wistar rats. (A) Ventral prostate of an untreated control rat. (B) Proliferation of the epithelium in the ventral prostate of a WIRS-treated rat, characterized by intraluminal villous enfolding, as well as the appearance of epithelial nodules, piling up of epithelial cells and a loss of cell polarity. NGF expression is increased. (C) Ventral prostate of a 6-OHDA-treated rat showing dilated acini and a significant increase in NGF level. (D) Ventral prostate of a WIRS plus 6-OHDA-treated rat without proliferation. (E) Negative control. All images were captured using the same magnification (original magnification, ×200; bar, 100 μm) and stained using Hematoxylin-Eosin. NGF, nerve growth factor; WIRS, restraint water-immersion stress; 6-OHDA, 6-hydroxydopamine.

Techniques Used: Expressing, Negative Control, Staining

38) Product Images from "Mitomycin-treated undifferentiated embryonic stem cells as a safe and effective therapeutic strategy in a mouse model of Parkinson’s disease"

Article Title: Mitomycin-treated undifferentiated embryonic stem cells as a safe and effective therapeutic strategy in a mouse model of Parkinson’s disease

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2015.00097

Transplantation of fully undifferentiated mESCs previously treated or non-treated with MMC into 6-OHDA-lesioned mice brains. (A) Survival curve of control and transplanted 6-OHDA-lesioned mice: 4 weeks after 6-OHDA injection in the striatum, mice were injected at the same stereotaxic coordinates with culture medium (lesion-control, n = 8), untreated mESCs (mESC, n = 8), or mitomycin-treated mESCs (MMC- mESC, n = 12). (B,C) In vivo brain MRI, coronal plane, T1-weighted sequence, prior to (left-hand panel) and after (right-hand panels) gadolinium injection evidencing striatal tumor 4 weeks after transplantation of untreated mESCs (B) and showing no signs of tumor formation in an animal transplanted with 500,000 mitomycin-treated mESCs for as long as 60 weeks (C). (D) Computer-microscope reconstruction of a coronal section through the forebrain of a 6-OHDA-lesioned mouse 4 weeks after transplantation with untreated mESCs into the striatum (dots indicate cell bodies positive for TH-immunostaining). (E) Histology of the boxed area in the computer-generated plot in (D) ; boxed areas in E are shown at higher magnification (F,G). (F) non-lesioned, non-transplanted side showing control level of TH staining (brown) in the striatum. (G) 6-OHDA-lesioned and mESC-grafted hemisphere, illustrating TH-positive cell bodies within the host striatum in close proximity to the tumor edges. Scale bar: 1000 µm and 1800 µm, respectively, for (D) , 500 µm for (E) , 100 µm for (F,G) .
Figure Legend Snippet: Transplantation of fully undifferentiated mESCs previously treated or non-treated with MMC into 6-OHDA-lesioned mice brains. (A) Survival curve of control and transplanted 6-OHDA-lesioned mice: 4 weeks after 6-OHDA injection in the striatum, mice were injected at the same stereotaxic coordinates with culture medium (lesion-control, n = 8), untreated mESCs (mESC, n = 8), or mitomycin-treated mESCs (MMC- mESC, n = 12). (B,C) In vivo brain MRI, coronal plane, T1-weighted sequence, prior to (left-hand panel) and after (right-hand panels) gadolinium injection evidencing striatal tumor 4 weeks after transplantation of untreated mESCs (B) and showing no signs of tumor formation in an animal transplanted with 500,000 mitomycin-treated mESCs for as long as 60 weeks (C). (D) Computer-microscope reconstruction of a coronal section through the forebrain of a 6-OHDA-lesioned mouse 4 weeks after transplantation with untreated mESCs into the striatum (dots indicate cell bodies positive for TH-immunostaining). (E) Histology of the boxed area in the computer-generated plot in (D) ; boxed areas in E are shown at higher magnification (F,G). (F) non-lesioned, non-transplanted side showing control level of TH staining (brown) in the striatum. (G) 6-OHDA-lesioned and mESC-grafted hemisphere, illustrating TH-positive cell bodies within the host striatum in close proximity to the tumor edges. Scale bar: 1000 µm and 1800 µm, respectively, for (D) , 500 µm for (E) , 100 µm for (F,G) .

Techniques Used: Transplantation Assay, Mouse Assay, Injection, In Vivo, Magnetic Resonance Imaging, Sequencing, Microscopy, Immunostaining, Generated, Staining

Functional recovery after transplantation of fully undifferentiated mESCs treated with MMC. (A) Rotational behavior induced by apomorphine (0.5 mg/kg i.p.) in 6-OHDA-lesioned mice. Eight weeks after the 6-OHDA lesion, mice received intra-striatal injections of culture medium (lesion-control, black), untreated 500,000 mESCs (blue), or 500,000 mitomycin-treated mESCs (MMC-mESC, red). After 12 weeks, all animals transplanted with mitomycin-treated mESCs were alive and had greatly reduced signs of striatal lesion, approaching the performances of normal healthy animals in the different tasks. Motor coordination on the balance beam test was assessed by the number of slips (B) and the latency to cross (C) a 50 cm long beam. (D) Spontaneous locomotor activity in the open field was tracked and expressed as the distance covered within 5 min. (E) Forelimb grip strength was measured as the maximal pull force on a bar. (F) Forelimb use during vertical exploratory behavior was measured in the cylinder test, in which normal animals most often touched the cylinder wall with both forepaws when rearing. Values are means ± SD. In (A) , (*) denotes p
Figure Legend Snippet: Functional recovery after transplantation of fully undifferentiated mESCs treated with MMC. (A) Rotational behavior induced by apomorphine (0.5 mg/kg i.p.) in 6-OHDA-lesioned mice. Eight weeks after the 6-OHDA lesion, mice received intra-striatal injections of culture medium (lesion-control, black), untreated 500,000 mESCs (blue), or 500,000 mitomycin-treated mESCs (MMC-mESC, red). After 12 weeks, all animals transplanted with mitomycin-treated mESCs were alive and had greatly reduced signs of striatal lesion, approaching the performances of normal healthy animals in the different tasks. Motor coordination on the balance beam test was assessed by the number of slips (B) and the latency to cross (C) a 50 cm long beam. (D) Spontaneous locomotor activity in the open field was tracked and expressed as the distance covered within 5 min. (E) Forelimb grip strength was measured as the maximal pull force on a bar. (F) Forelimb use during vertical exploratory behavior was measured in the cylinder test, in which normal animals most often touched the cylinder wall with both forepaws when rearing. Values are means ± SD. In (A) , (*) denotes p

Techniques Used: Functional Assay, Transplantation Assay, Mouse Assay, Activity Assay

39) Product Images from "Constitutive translation of human α-synuclein is mediated by the 5′-untranslated region"

Article Title: Constitutive translation of human α-synuclein is mediated by the 5′-untranslated region

Journal: Open Biology

doi: 10.1098/rsob.160022

The SNCA IRES activity is induced by different stress signals. HEK-293 cells were transiently transfected with SNCA or GAPDH pRF plasmids. Four hours later, cells were treated with different stress stimuli. Forty-eight hours later, cells were harvested and dual luciferase assays were performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla (Fluc/Rluc) activity. Note that KCl, FeSO 4 , 6-OHDA, serum deprivation but not MPP + treatment induced SNCA IRES activity. The figure represents averages and standard errors of four independent experiments. ** p
Figure Legend Snippet: The SNCA IRES activity is induced by different stress signals. HEK-293 cells were transiently transfected with SNCA or GAPDH pRF plasmids. Four hours later, cells were treated with different stress stimuli. Forty-eight hours later, cells were harvested and dual luciferase assays were performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla (Fluc/Rluc) activity. Note that KCl, FeSO 4 , 6-OHDA, serum deprivation but not MPP + treatment induced SNCA IRES activity. The figure represents averages and standard errors of four independent experiments. ** p

Techniques Used: Activity Assay, Transfection, Luciferase

The SNCA 5′-UTR enhances SNCA ORF translation during stress. Freshly plated HEK-293 cells were either serum-deprived (w/o FBS) or treated with different stressors (KCl, ferrous ammonium, 6-OHDA and MPP + ). Forty-eight hours later, cells were harvested and SNCA protein ( a ) and mRNA ( b ) levels were determined and normalized to GAPDH following immunoblotting and real-time RT-PCR, respectively. Note that all treatments induced SNCA protein levels. From these, 6-OHDA and serum deprivation also induced SNCA mRNA levels indicating that their effect on SNCA protein levels may, in part, be mediated by enhanced mRNA expression. ( c,d ) The SNCA pcDNA3.1 5′-CDS-3′ and kozac-CDS-3′ plasmids were transiently co-transfected into Neuro-2a cells with DNA6.2-GW/EmGFP plasmid which served as an internal control of transfection. Four hours later, cells were treated with different stress stimuli. Forty-eight hours later, cells were harvested and SNCA protein and mRNA levels were determined and normalized to EmGFP levels following immunoblotting and real-time RT-PCR, respectively. Note that SNCA 5′-UTR promoted translation under different stress conditions. The figures represent averages and standard errors of four to six independent experiments. * p
Figure Legend Snippet: The SNCA 5′-UTR enhances SNCA ORF translation during stress. Freshly plated HEK-293 cells were either serum-deprived (w/o FBS) or treated with different stressors (KCl, ferrous ammonium, 6-OHDA and MPP + ). Forty-eight hours later, cells were harvested and SNCA protein ( a ) and mRNA ( b ) levels were determined and normalized to GAPDH following immunoblotting and real-time RT-PCR, respectively. Note that all treatments induced SNCA protein levels. From these, 6-OHDA and serum deprivation also induced SNCA mRNA levels indicating that their effect on SNCA protein levels may, in part, be mediated by enhanced mRNA expression. ( c,d ) The SNCA pcDNA3.1 5′-CDS-3′ and kozac-CDS-3′ plasmids were transiently co-transfected into Neuro-2a cells with DNA6.2-GW/EmGFP plasmid which served as an internal control of transfection. Four hours later, cells were treated with different stress stimuli. Forty-eight hours later, cells were harvested and SNCA protein and mRNA levels were determined and normalized to EmGFP levels following immunoblotting and real-time RT-PCR, respectively. Note that SNCA 5′-UTR promoted translation under different stress conditions. The figures represent averages and standard errors of four to six independent experiments. * p

Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation

40) Product Images from "Botulinum Neurotoxin-A Injected Intrastriatally into Hemiparkinsonian Rats Improves the Initiation Time for Left and Right Forelimbs in Both Forehand and Backhand Directions"

Article Title: Botulinum Neurotoxin-A Injected Intrastriatally into Hemiparkinsonian Rats Improves the Initiation Time for Left and Right Forelimbs in Both Forehand and Backhand Directions

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20040992

Line/scatter plots of the linear regression of the initiation time of the left ( A , B ) and right ( C , D ) forepaws in both forehand ( A , C ) and backhand ( B , D ) directions in sham BoNT-A-injected hemi-PD rats during post-lesion experimental period. The open circles represent the initiation times plotted against the time after the 6-OHDA lesion, the solid black line running through the points represents the regression line, and the colored lines represent the prediction and confidence intervals. The confidence interval for the population (prediction interval, red lines) gives the range of values that define the region containing the population from which the observations were drawn. The confidence interval for the regression line (blue lines) gives the range of values that defines the region containing the true mean relationship between the dependent and independent variables, with the specified level (95%) of confidence.
Figure Legend Snippet: Line/scatter plots of the linear regression of the initiation time of the left ( A , B ) and right ( C , D ) forepaws in both forehand ( A , C ) and backhand ( B , D ) directions in sham BoNT-A-injected hemi-PD rats during post-lesion experimental period. The open circles represent the initiation times plotted against the time after the 6-OHDA lesion, the solid black line running through the points represents the regression line, and the colored lines represent the prediction and confidence intervals. The confidence interval for the population (prediction interval, red lines) gives the range of values that define the region containing the population from which the observations were drawn. The confidence interval for the regression line (blue lines) gives the range of values that defines the region containing the true mean relationship between the dependent and independent variables, with the specified level (95%) of confidence.

Techniques Used: Injection

Correlation analysis (Pearson) of the initiation time of stepping movements of the left forepaw (contralateral to the 6-OHDA lesion) in the forehand direction and apomorphine-induced rotations in hemi-PD rats injected intrastriatally with BoNT-A (closed circles) or sham BoNT-A (open circles). No significant correlations between the ITs of the left forepaw in the forehand direction and apomorphine-induced rotations were found 1 month ( A ), 3 months ( C ), and 6 months ( E ) after the first BoNT-A or 1 month ( B ), 3 months ( D ), and 6 months ( F ) after the second BoNT-A. Data represent means ± SEM. Linear regression lines are displayed for BoNT (solid) and Sham (dotted) groups.
Figure Legend Snippet: Correlation analysis (Pearson) of the initiation time of stepping movements of the left forepaw (contralateral to the 6-OHDA lesion) in the forehand direction and apomorphine-induced rotations in hemi-PD rats injected intrastriatally with BoNT-A (closed circles) or sham BoNT-A (open circles). No significant correlations between the ITs of the left forepaw in the forehand direction and apomorphine-induced rotations were found 1 month ( A ), 3 months ( C ), and 6 months ( E ) after the first BoNT-A or 1 month ( B ), 3 months ( D ), and 6 months ( F ) after the second BoNT-A. Data represent means ± SEM. Linear regression lines are displayed for BoNT (solid) and Sham (dotted) groups.

Techniques Used: Injection

Scatter plots of the initiation time ( A ) and adjusting steps ( B ) in stepping test and apomorphine-induced rotational behavior 1 month after the 6-OHDA lesion. ( A ) No statistically significant correlation between the initiation time of movements of both forelimbs in both forehand and backhand directions and apomorphine-induced rotations after 1 month following the 6-OHDA lesion was found. ( B ) Comparing the adjusting steps for the both forelimbs in forehand and backhand directions with apomorphine-induced rotations 1 month following the 6-OHDA lesion showed no significant correlations. Linear regression lines are displayed for BoNT (solid) and Sham (dotted) groups. For better readability of the figure no error bars are shown.
Figure Legend Snippet: Scatter plots of the initiation time ( A ) and adjusting steps ( B ) in stepping test and apomorphine-induced rotational behavior 1 month after the 6-OHDA lesion. ( A ) No statistically significant correlation between the initiation time of movements of both forelimbs in both forehand and backhand directions and apomorphine-induced rotations after 1 month following the 6-OHDA lesion was found. ( B ) Comparing the adjusting steps for the both forelimbs in forehand and backhand directions with apomorphine-induced rotations 1 month following the 6-OHDA lesion showed no significant correlations. Linear regression lines are displayed for BoNT (solid) and Sham (dotted) groups. For better readability of the figure no error bars are shown.

Techniques Used:

Initiation time of stepping behavior in right side hemiparkinsonian (hemi-PD) rats treated with repetitive intrastriatal BoNT-A or vehicle. Before the 6-OHDA lesion, rats of both experimental groups needed about 0.62 s to start a movement with their left ( A , B ) and right ( C , D ) forepaws in both forehand and backhand directions. After the 6-OHDA lesion the initiation time in hemi-PD rats was significantly increased in the left forepaw (contralateral to lesion) in both forehand ( A ) and backhand ( B ) directions. Unilateral intrastriatal BoNT-A injection significantly decreased the initiation time of the left forepaw in both forehand ( A ) and backhand ( B ) directions as well as 1 month and 3 months after 1. BoNT-A injection and 1 month after the 2. BoNT-A injection compared to the sham-injected group. The initiation time of movement in the right forepaw in hemi-PD rats in both forehand ( C ) and backhand ( D ) directions were not affected 1 month after lesion nor by the first or second ipsilateral BoNT-A or vehicle injections. Asterisks indicate significant differences compared with the sham group (*** p
Figure Legend Snippet: Initiation time of stepping behavior in right side hemiparkinsonian (hemi-PD) rats treated with repetitive intrastriatal BoNT-A or vehicle. Before the 6-OHDA lesion, rats of both experimental groups needed about 0.62 s to start a movement with their left ( A , B ) and right ( C , D ) forepaws in both forehand and backhand directions. After the 6-OHDA lesion the initiation time in hemi-PD rats was significantly increased in the left forepaw (contralateral to lesion) in both forehand ( A ) and backhand ( B ) directions. Unilateral intrastriatal BoNT-A injection significantly decreased the initiation time of the left forepaw in both forehand ( A ) and backhand ( B ) directions as well as 1 month and 3 months after 1. BoNT-A injection and 1 month after the 2. BoNT-A injection compared to the sham-injected group. The initiation time of movement in the right forepaw in hemi-PD rats in both forehand ( C ) and backhand ( D ) directions were not affected 1 month after lesion nor by the first or second ipsilateral BoNT-A or vehicle injections. Asterisks indicate significant differences compared with the sham group (*** p

Techniques Used: Injection

Initiation time of movements ( A ) and adjusting steps ( B ) for the left and right forepaws in stepping test measured 1 month after 6-OHDA injection into the right MFB. ( A ) One month after the 6-OHDA lesion the IT of movements of the left forelimb in both forehand and backhand directions were significantly increased compared to the right forelimb. ( B ) The right side 6-OHDA lesion induced impairments of the adjusting steps of the left forelimb in both forehand and backhand directions. Asterisks indicate significant differences (* p
Figure Legend Snippet: Initiation time of movements ( A ) and adjusting steps ( B ) for the left and right forepaws in stepping test measured 1 month after 6-OHDA injection into the right MFB. ( A ) One month after the 6-OHDA lesion the IT of movements of the left forelimb in both forehand and backhand directions were significantly increased compared to the right forelimb. ( B ) The right side 6-OHDA lesion induced impairments of the adjusting steps of the left forelimb in both forehand and backhand directions. Asterisks indicate significant differences (* p

Techniques Used: Injection

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Mutagenesis:

Article Title: Oxidation of Survival Factor MEF2D in Neuronal Death and Parkinson's Disease
Article Snippet: .. The OxyBlot protein oxidation detection kit was purchased from Chemicon International (S7150), QuikChange site-directed mutagenesis kit from Stratagene (200522), TRIzol from Invitrogen (15596-026), TURBO DNA-free™ kit from Ambion (AM1907), SYBR Green Brilliant III Ultra-fast reagent kit from Applied Biosystems (6008223), 6-OHDA from Sigma (H8523), Biotin-iodoacetamide from Invitrogen (B1591), iodoacetamide (IAM) and α-tocopherol from Sigma (I6125), and MTT Kit I from Boehringer Mannheim (1465007). .. The Cytotoxicity LDH Detection Kit was purchased from Roche (04744926001).

Concentration Assay:

Article Title: Neuroprotective Activity of Some Marine Fungal Metabolites in the 6-Hydroxydopamin- and Paraquat-Induced Parkinson’s Disease Models
Article Snippet: .. Neuroblastoma Neuro2a line cells (1 × 103 cells/well) were treated with 50 µM of 6-hydroxydopamine (Sigma-Aldrich, St. Louis, MO, USA) for 1 h and, after that, the investigated compounds were added to the neuroblastoma cell suspension at a concentration of 1 and 10 µM. .. Cells incubated without 6-OHDA and compounds, and with 6-OHDA only, were used as positive and negative controls, respectively.

Injection:

Article Title: The Effect of Catecholaminergic Depletion Within the Prelimbic and Infralimbic Medial Prefrontal Cortex on Recognition Memory for Recency, Location, and Objects
Article Snippet: .. 6-OHDA hydrobromide (24 mg/ml as salt dissolved in vehicle; Sigma, United Kingdom) or vehicle (0.9% saline/ascorbic acid 0.01% wt/vol) was infused manually over 2 min bilaterally in a volume of 0.2 μl per injection site. ..

Article Title: Treatment of Parkinson's disease: nanostructured sol-gel silica-dopamine reservoirs for controlled drug release in the central nervous system
Article Snippet: .. A single dose of 6-hydroxydopamine 9 μg/3 μL, (Sigma-Aldrich) was manually injected in small steps (at approximately 0.2 μL/min) into the right substantia nigra pars compacta, at the coordinates AP , −5.3 mm from bregma; L , −1.8 mm from the midline, and V , −7.6 mm from the dura surface. ..

SYBR Green Assay:

Article Title: Oxidation of Survival Factor MEF2D in Neuronal Death and Parkinson's Disease
Article Snippet: .. The OxyBlot protein oxidation detection kit was purchased from Chemicon International (S7150), QuikChange site-directed mutagenesis kit from Stratagene (200522), TRIzol from Invitrogen (15596-026), TURBO DNA-free™ kit from Ambion (AM1907), SYBR Green Brilliant III Ultra-fast reagent kit from Applied Biosystems (6008223), 6-OHDA from Sigma (H8523), Biotin-iodoacetamide from Invitrogen (B1591), iodoacetamide (IAM) and α-tocopherol from Sigma (I6125), and MTT Kit I from Boehringer Mannheim (1465007). .. The Cytotoxicity LDH Detection Kit was purchased from Roche (04744926001).

MTT Assay:

Article Title: Protective effects of flavonol isoquercitrin, against 6-hydroxy dopamine (6-OHDA) - induced toxicity in PC12 cells
Article Snippet: .. 6-hydroxydopamine, isoquercitrin, poly-L-Lysine, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide),3,4 – dihydroxy-L-phenylalanine (Levodopa) and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich (USA). .. Dulbecco’s modified eagle’s medium (DMEM), pen-strep, horse serum, and fetal bovine serum was purchased from GIBCO Inc. (USA).

Article Title: Oxidation of Survival Factor MEF2D in Neuronal Death and Parkinson's Disease
Article Snippet: .. The OxyBlot protein oxidation detection kit was purchased from Chemicon International (S7150), QuikChange site-directed mutagenesis kit from Stratagene (200522), TRIzol from Invitrogen (15596-026), TURBO DNA-free™ kit from Ambion (AM1907), SYBR Green Brilliant III Ultra-fast reagent kit from Applied Biosystems (6008223), 6-OHDA from Sigma (H8523), Biotin-iodoacetamide from Invitrogen (B1591), iodoacetamide (IAM) and α-tocopherol from Sigma (I6125), and MTT Kit I from Boehringer Mannheim (1465007). .. The Cytotoxicity LDH Detection Kit was purchased from Roche (04744926001).

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    Millipore 6 hydroxydopamine hydrochloride
    Functions of the LEV-1 nAChR subunit in DNs. (A) lev-1(lf) caused basal slowing deficits when compared wild-type animals as shown by increased BSR index (% body bends per 20 seconds on food per animal relative to average body bends per 20 seconds off food in the same experiment for wild-type, N, (N=2, n=30 each) or lev-1(lf) (N=2, n=15 each) animals). (B) lev-1(lf) animals had increased sensitivity to the paralyzing effects of exogenous dopamine. Results depicted as % animals paralyzed after 20 minutes in the presence of exogenous dopamine, 6 plates, 10 animals per plate. (C) lev-1(lf) abolishes nicotine-induced protection against <t>6-OHDA</t> toxicity. Effects of chronic nicotine exposure on 6-OHDA-induced DN degeneration in lev-1(lf); P dat-1 ::GFP ( vtIs7 ) animals expressing GFP in their DNs is compared to effects of this treatment on the control BY250 strain [P dat-1 ::GFP ( vtIs7 )] animals expressing GFP only in DNs N=3, n=30 each. Data represented as average + SD; significance was examined using two-tailed unpaired Student’s t -test (A, B) or one-way ANOVA with Tukey’s post hoc multiple comparisons (C); ns - p > 0.05, *- p = 0.0149, **- p
    6 Hydroxydopamine Hydrochloride, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ECH protected PC1 2 from <t>6‐OHDA‐induced</t> ERS through promoting seipin ubiquitination and degradation. ( A ) ECH down‐regulated seipin and ERS‐associated protein expression induced by 6‐OHDA. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 3, 40 μg protein each lane). ( B ) Knocking down seipin by siRNA attenuated ERS‐associated protein accumulation induced by 6‐OHDA. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 3, 40 μg protein each lane). SiRNA, 50 nM each group transfected and collected to obtain protein lysis after 48 hrs. ( C ) The relative mRNA levels of seipin were determined using RT‐PCR. The results of RT‐PCR were normalized to GAPDH and expressed as fold change to control. SiRNA of seipin dramatically inhibited mRNA levels of seipin ( P
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    Millipore 6 ohda hbr
    Using photometry to measure D1R-dependent PKA activation in intact and <t>6-OHDA</t> lesioned hemi-Parkinsonian rats. Panels a and b show cylinder test results for intact rats and 6-OHDA lesioned rats, respectively. Rats in both groups had been implanted with optic probes and expressed AKAR in the dorsal striatum, in the case of 6-OHDA injected animals this was ipsilateral to the lesion. Y-axes show the percentage of rears in which individual animals used the forelimb that was ipsilateral vs . contralateral to the AAV-injected hemisphere, or used both forelimbs at the same time. Tests in the 6-OHDA lesioned rats occurred 3 weeks following intra-striatal injection of AAV 1 -SynTet-AKAR and optical probe implantation. (c) Representative immunofluorescent images depicting AKAR expression (green) and optic-probe placement in the dorsal striatum in addition to expression of tyrosine hydroxylase (TH, shown in purple) in the striatum, medial forebrain bundle (MFB) and substantia nigra (SN). (d) FRET recordings performed on intact rats expressing AKAR and injected with either saline or the D1R agonist SKF 81297 (0.5 mg/kg s.c.) at t = 10 min (n = 12). (e) FRET recordings performed on 6-OHDA lesioned rats expressing AKAR and injected with either saline or SKF81297 (0.5 mg/kg s.c.) at t = 10 min (n = 9). For all experiments, rats were recorded under both saline and drug conditions in a counterbalanced design. (f) Area under the curve (AUC) analysis of panels d and e. AUC was calculated relative to baseline following drug or saline injection. AUC values for Saline vs . SKF 81297 in each control and lesioned rats were by repeated measures t-tests, * p
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    Functions of the LEV-1 nAChR subunit in DNs. (A) lev-1(lf) caused basal slowing deficits when compared wild-type animals as shown by increased BSR index (% body bends per 20 seconds on food per animal relative to average body bends per 20 seconds off food in the same experiment for wild-type, N, (N=2, n=30 each) or lev-1(lf) (N=2, n=15 each) animals). (B) lev-1(lf) animals had increased sensitivity to the paralyzing effects of exogenous dopamine. Results depicted as % animals paralyzed after 20 minutes in the presence of exogenous dopamine, 6 plates, 10 animals per plate. (C) lev-1(lf) abolishes nicotine-induced protection against 6-OHDA toxicity. Effects of chronic nicotine exposure on 6-OHDA-induced DN degeneration in lev-1(lf); P dat-1 ::GFP ( vtIs7 ) animals expressing GFP in their DNs is compared to effects of this treatment on the control BY250 strain [P dat-1 ::GFP ( vtIs7 )] animals expressing GFP only in DNs N=3, n=30 each. Data represented as average + SD; significance was examined using two-tailed unpaired Student’s t -test (A, B) or one-way ANOVA with Tukey’s post hoc multiple comparisons (C); ns - p > 0.05, *- p = 0.0149, **- p

    Journal: bioRxiv

    Article Title: Conserved nicotine-activated neuroprotective pathways involve mitochondrial stress

    doi: 10.1101/2020.05.28.120576

    Figure Lengend Snippet: Functions of the LEV-1 nAChR subunit in DNs. (A) lev-1(lf) caused basal slowing deficits when compared wild-type animals as shown by increased BSR index (% body bends per 20 seconds on food per animal relative to average body bends per 20 seconds off food in the same experiment for wild-type, N, (N=2, n=30 each) or lev-1(lf) (N=2, n=15 each) animals). (B) lev-1(lf) animals had increased sensitivity to the paralyzing effects of exogenous dopamine. Results depicted as % animals paralyzed after 20 minutes in the presence of exogenous dopamine, 6 plates, 10 animals per plate. (C) lev-1(lf) abolishes nicotine-induced protection against 6-OHDA toxicity. Effects of chronic nicotine exposure on 6-OHDA-induced DN degeneration in lev-1(lf); P dat-1 ::GFP ( vtIs7 ) animals expressing GFP in their DNs is compared to effects of this treatment on the control BY250 strain [P dat-1 ::GFP ( vtIs7 )] animals expressing GFP only in DNs N=3, n=30 each. Data represented as average + SD; significance was examined using two-tailed unpaired Student’s t -test (A, B) or one-way ANOVA with Tukey’s post hoc multiple comparisons (C); ns - p > 0.05, *- p = 0.0149, **- p

    Article Snippet: Animals were placed back onto nicotine containing plates post-6-OHDA treatments; 2) for acute nicotine tests, animals were only exposed to nicotine while in the 6-OHDA assay mix; 3) for pre-exposure, a one-hour nicotine treatment was done 24-four hours prior to 6-OHDA, in addition to nicotine being present in the 6-OHDA assay mix.

    Techniques: Expressing, Two Tailed Test

    Conserved mechanisms mediate protective effects of nicotine. (A) Timeline depicting RNAi and chronic nicotine treatments. Animals were raised on RNAi bacteria for two generations (P0 and F1). Only the F1 generation were exposed to nicotine. (B-D) RNAi experiments involving the knockdown of ric-3 or egl-19 (B) or tax-6 (C) or dop-2 (D) in the DN-specific RNAi-sensitive strain, UA202, when animals were grown in the presence (grey bars) or absence (black bars) of 62μM nicotine and exposed to 10mM 6-OHDA/2mM ascorbic acid. (B) egl-19 and (C) tax-6 knockdown alone protected DNs from 6-OHDA toxicity and nicotine had no additional effects. (D) dop-2 knockdown abolished nicotine-induced protection from 6-OHDA. Data shown as average + SD (N=3, n= 30 animals each). Significance was examined using two-way ANOVA with Tukey’s post hoc multiple comparison test; ns - p > 0.05, **** - p

    Journal: bioRxiv

    Article Title: Conserved nicotine-activated neuroprotective pathways involve mitochondrial stress

    doi: 10.1101/2020.05.28.120576

    Figure Lengend Snippet: Conserved mechanisms mediate protective effects of nicotine. (A) Timeline depicting RNAi and chronic nicotine treatments. Animals were raised on RNAi bacteria for two generations (P0 and F1). Only the F1 generation were exposed to nicotine. (B-D) RNAi experiments involving the knockdown of ric-3 or egl-19 (B) or tax-6 (C) or dop-2 (D) in the DN-specific RNAi-sensitive strain, UA202, when animals were grown in the presence (grey bars) or absence (black bars) of 62μM nicotine and exposed to 10mM 6-OHDA/2mM ascorbic acid. (B) egl-19 and (C) tax-6 knockdown alone protected DNs from 6-OHDA toxicity and nicotine had no additional effects. (D) dop-2 knockdown abolished nicotine-induced protection from 6-OHDA. Data shown as average + SD (N=3, n= 30 animals each). Significance was examined using two-way ANOVA with Tukey’s post hoc multiple comparison test; ns - p > 0.05, **** - p

    Article Snippet: Animals were placed back onto nicotine containing plates post-6-OHDA treatments; 2) for acute nicotine tests, animals were only exposed to nicotine while in the 6-OHDA assay mix; 3) for pre-exposure, a one-hour nicotine treatment was done 24-four hours prior to 6-OHDA, in addition to nicotine being present in the 6-OHDA assay mix.

    Techniques:

    Chronic nicotine treatment protects DNs in a RIC-3 dependent manner. (A) Timeline of experiments. (A-Top) Control experiments without nicotine exposure. (A-Bottom) Animals in nicotine group exposed to 62μM nicotine from hatching, during and after 6-OHDA exposure. Degeneration of DNs was scored twenty-four hours after 10mM 6-OHDA/2mM ascorbic acid exposure or 2mM ascorbic acid alone. (B) Chronic nicotine treatment attenuates 6-OHDA-induced DN degeneration in BY250 worms expressing only GFP in their DNs [P dat-1:: GFP ( vtIs7 )]. (C-F) Representative images matching the conditions of panel B. (C) Solvent (ddH 2 O/2mM ascorbic acid) control with all six anterior DNs intact (arrowheads). (D) Nicotine only treatment with all six anterior DNs intact (arrowheads). (E) 6-OHDA-induced DN degeneration, with three degenerated (arrows) and three intact (arrowheads) DNs depicted. (F) Nicotine treatment of 6-OHDA treated animals with all six anterior DNs intact and protected from 6-OHDA toxicity (arrowheads). (G) DN-specific RNAi knockdown of ric-3 (using UA202 worms) abolishes nicotine-induced protection against 6-OHDA toxicity compared to empty vector (EV) control. Data is represented as average + SD (N=3, n= 30 each). Significance was examined using two-way ANOVA with Tukey’s post hoc multiple comparison test (A, F); ns - p > 0.05, *** - p

    Journal: bioRxiv

    Article Title: Conserved nicotine-activated neuroprotective pathways involve mitochondrial stress

    doi: 10.1101/2020.05.28.120576

    Figure Lengend Snippet: Chronic nicotine treatment protects DNs in a RIC-3 dependent manner. (A) Timeline of experiments. (A-Top) Control experiments without nicotine exposure. (A-Bottom) Animals in nicotine group exposed to 62μM nicotine from hatching, during and after 6-OHDA exposure. Degeneration of DNs was scored twenty-four hours after 10mM 6-OHDA/2mM ascorbic acid exposure or 2mM ascorbic acid alone. (B) Chronic nicotine treatment attenuates 6-OHDA-induced DN degeneration in BY250 worms expressing only GFP in their DNs [P dat-1:: GFP ( vtIs7 )]. (C-F) Representative images matching the conditions of panel B. (C) Solvent (ddH 2 O/2mM ascorbic acid) control with all six anterior DNs intact (arrowheads). (D) Nicotine only treatment with all six anterior DNs intact (arrowheads). (E) 6-OHDA-induced DN degeneration, with three degenerated (arrows) and three intact (arrowheads) DNs depicted. (F) Nicotine treatment of 6-OHDA treated animals with all six anterior DNs intact and protected from 6-OHDA toxicity (arrowheads). (G) DN-specific RNAi knockdown of ric-3 (using UA202 worms) abolishes nicotine-induced protection against 6-OHDA toxicity compared to empty vector (EV) control. Data is represented as average + SD (N=3, n= 30 each). Significance was examined using two-way ANOVA with Tukey’s post hoc multiple comparison test (A, F); ns - p > 0.05, *** - p

    Article Snippet: Animals were placed back onto nicotine containing plates post-6-OHDA treatments; 2) for acute nicotine tests, animals were only exposed to nicotine while in the 6-OHDA assay mix; 3) for pre-exposure, a one-hour nicotine treatment was done 24-four hours prior to 6-OHDA, in addition to nicotine being present in the 6-OHDA assay mix.

    Techniques: Expressing, Plasmid Preparation

    Pre-exposure to nicotine protects DNs from 6-OHDA. (A) Timeline depicting nicotine treatments protocols. (A-Top) Control protocol. (A-Middle) Acute exposure, addition of 62μM to the assay mix only during 6-OHDA treatment. (A-Bottom) Pre-exposure, one-hour pre-exposure to 62μM nicotine the day before 6-OHDA + nicotine (acute exposure). Examined in BY250 worms expressing GFP only in DNs. (B) Acute nicotine is not neuroprotective. (C) A nicotine pre-exposure + acute exposure is protective against 6-OHDA toxicity. Data represented as average + SD (N=3, n=30 worms each). Significance was examined using two-tailed unpaired Student’s t -test; ns - p > 0.05, * - p= 0.0178.

    Journal: bioRxiv

    Article Title: Conserved nicotine-activated neuroprotective pathways involve mitochondrial stress

    doi: 10.1101/2020.05.28.120576

    Figure Lengend Snippet: Pre-exposure to nicotine protects DNs from 6-OHDA. (A) Timeline depicting nicotine treatments protocols. (A-Top) Control protocol. (A-Middle) Acute exposure, addition of 62μM to the assay mix only during 6-OHDA treatment. (A-Bottom) Pre-exposure, one-hour pre-exposure to 62μM nicotine the day before 6-OHDA + nicotine (acute exposure). Examined in BY250 worms expressing GFP only in DNs. (B) Acute nicotine is not neuroprotective. (C) A nicotine pre-exposure + acute exposure is protective against 6-OHDA toxicity. Data represented as average + SD (N=3, n=30 worms each). Significance was examined using two-tailed unpaired Student’s t -test; ns - p > 0.05, * - p= 0.0178.

    Article Snippet: Animals were placed back onto nicotine containing plates post-6-OHDA treatments; 2) for acute nicotine tests, animals were only exposed to nicotine while in the 6-OHDA assay mix; 3) for pre-exposure, a one-hour nicotine treatment was done 24-four hours prior to 6-OHDA, in addition to nicotine being present in the 6-OHDA assay mix.

    Techniques: Expressing, Two Tailed Test

    Protective effects of nicotine and mitochondrial stress. (A) A hsp-6::GFP reporter strain chronically treated with 62μM nicotine showed increased mitochondrial stress (N=2-3, n=30 each). (B-E) RNAi experiments knocking down gene targets in the DNs of selectively RNAi-sensitive UA202 animals grown in the presence (grey bars) or absence (black bars) of 62μM nicotine and exposed to10mM 6-OHDA/2mM ascorbic acid (N= 3, n=30 each). (B) mcu-1 knockdown abolished nicotine-induced protection. (C) micu-1 knockdown had no effect on nicotine-induced protection. (D) atfs-1 knockdown alone protects against 6-OHDA toxicity, while the addition of nicotine exposure decreased its protective effects. (E) pink-1 knockdown abolished nicotine-induced protection. Data shown as average + SD and significance was examined using two-tailed t-test (A), or two-way ANOVA with Tukey’s post hoc multiple comparison test (B-E); ns - p > 0.05, *** - p

    Journal: bioRxiv

    Article Title: Conserved nicotine-activated neuroprotective pathways involve mitochondrial stress

    doi: 10.1101/2020.05.28.120576

    Figure Lengend Snippet: Protective effects of nicotine and mitochondrial stress. (A) A hsp-6::GFP reporter strain chronically treated with 62μM nicotine showed increased mitochondrial stress (N=2-3, n=30 each). (B-E) RNAi experiments knocking down gene targets in the DNs of selectively RNAi-sensitive UA202 animals grown in the presence (grey bars) or absence (black bars) of 62μM nicotine and exposed to10mM 6-OHDA/2mM ascorbic acid (N= 3, n=30 each). (B) mcu-1 knockdown abolished nicotine-induced protection. (C) micu-1 knockdown had no effect on nicotine-induced protection. (D) atfs-1 knockdown alone protects against 6-OHDA toxicity, while the addition of nicotine exposure decreased its protective effects. (E) pink-1 knockdown abolished nicotine-induced protection. Data shown as average + SD and significance was examined using two-tailed t-test (A), or two-way ANOVA with Tukey’s post hoc multiple comparison test (B-E); ns - p > 0.05, *** - p

    Article Snippet: Animals were placed back onto nicotine containing plates post-6-OHDA treatments; 2) for acute nicotine tests, animals were only exposed to nicotine while in the 6-OHDA assay mix; 3) for pre-exposure, a one-hour nicotine treatment was done 24-four hours prior to 6-OHDA, in addition to nicotine being present in the 6-OHDA assay mix.

    Techniques: Two Tailed Test

    Overexpressed HPRT1 activates the Wnt/β-catenin signaling pathway in a PD model. 6-OHDA-treated N27 dopaminergic neurons were treated with oe-NC or oe-HPRT1. ( A ) The protein expression of total-β-catenin and DAT as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio using TOP/FOP flash reporter assay. FOP was designated as background value or as negative control due to its stability. * p

    Journal: Aging (Albany NY)

    Article Title: LncRNA H19 diminishes dopaminergic neuron loss by mediating microRNA-301b-3p in Parkinson’s disease via the HPRT1-mediated Wnt/β-catenin signaling pathway

    doi: 10.18632/aging.102877

    Figure Lengend Snippet: Overexpressed HPRT1 activates the Wnt/β-catenin signaling pathway in a PD model. 6-OHDA-treated N27 dopaminergic neurons were treated with oe-NC or oe-HPRT1. ( A ) The protein expression of total-β-catenin and DAT as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio using TOP/FOP flash reporter assay. FOP was designated as background value or as negative control due to its stability. * p

    Article Snippet: ImmunohistochemistryAt day 14 after administration of 6-OHDA, the mice were anesthetized with pentobarbital sodium.

    Techniques: Expressing, Western Blot, Activity Assay, Reporter Assay, Negative Control

    Schematic representation of H19 in regulating dopaminergic neuron loss in PD. H19 upregulates the expression of HPRT1 by binding to miR-301b-3p. Overexpression of HPRT1 could activate the Wnt/β-catenin signaling pathway, thus promoting the mRNA expression of Nurr-1, Pitx-3, Ngn-2 and NeuroD1 in the substantia nigra tissues, which ultimately rescues the dopaminergic neuron loss in this 6-OHDA-induced PD mouse model.

    Journal: Aging (Albany NY)

    Article Title: LncRNA H19 diminishes dopaminergic neuron loss by mediating microRNA-301b-3p in Parkinson’s disease via the HPRT1-mediated Wnt/β-catenin signaling pathway

    doi: 10.18632/aging.102877

    Figure Lengend Snippet: Schematic representation of H19 in regulating dopaminergic neuron loss in PD. H19 upregulates the expression of HPRT1 by binding to miR-301b-3p. Overexpression of HPRT1 could activate the Wnt/β-catenin signaling pathway, thus promoting the mRNA expression of Nurr-1, Pitx-3, Ngn-2 and NeuroD1 in the substantia nigra tissues, which ultimately rescues the dopaminergic neuron loss in this 6-OHDA-induced PD mouse model.

    Article Snippet: ImmunohistochemistryAt day 14 after administration of 6-OHDA, the mice were anesthetized with pentobarbital sodium.

    Techniques: Expressing, Binding Assay, Over Expression

    Overexpressed HPRT1 inhibits dopaminergic neuron loss in 6-OHDA-induced PD mice. 6-OHDA-induced PD mice were treated with oe-NC or oe-HPRT1. ( A ) Protein expression of HPRT1 in the substantia nigra tissues examined by western blot assay. ( B ) TH positive neurons in the substantia nigra examined by immunohistochemistry (upper × 100, lower × 400). ( C ) Fluoro-Jade B-stained apoptotic neurons (scale bar = 50 μm). ( D – G ) The mRNA expression of Nurr-1 ( D ), Pitx-3 ( E ), Ngn-2 ( F ) and NeuroD1 ( G ) in the substantia nigra tissues examined by RT-qPCR. * p

    Journal: Aging (Albany NY)

    Article Title: LncRNA H19 diminishes dopaminergic neuron loss by mediating microRNA-301b-3p in Parkinson’s disease via the HPRT1-mediated Wnt/β-catenin signaling pathway

    doi: 10.18632/aging.102877

    Figure Lengend Snippet: Overexpressed HPRT1 inhibits dopaminergic neuron loss in 6-OHDA-induced PD mice. 6-OHDA-induced PD mice were treated with oe-NC or oe-HPRT1. ( A ) Protein expression of HPRT1 in the substantia nigra tissues examined by western blot assay. ( B ) TH positive neurons in the substantia nigra examined by immunohistochemistry (upper × 100, lower × 400). ( C ) Fluoro-Jade B-stained apoptotic neurons (scale bar = 50 μm). ( D – G ) The mRNA expression of Nurr-1 ( D ), Pitx-3 ( E ), Ngn-2 ( F ) and NeuroD1 ( G ) in the substantia nigra tissues examined by RT-qPCR. * p

    Article Snippet: ImmunohistochemistryAt day 14 after administration of 6-OHDA, the mice were anesthetized with pentobarbital sodium.

    Techniques: Mouse Assay, Expressing, Western Blot, Immunohistochemistry, Staining, Quantitative RT-PCR

    Overexpressed HPRT1 inhibits dopaminergic neuron loss via the Wnt/β-catenin signaling pathway in 6-OHDA-treated N27 dopaminergic neurons and mice. N27 dopaminergic neurons and mice were treated with oe-HPRT1 in the presence of the Wnt/β-catenin blocker XAV-939 or DMSO carrier. ( A ) The protein expression of total-β-catenin, DAT and HPRT1 as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio in the N27 dopaminergic neurons. FOP was designated as background value or as negative control due to its stability. ( C ) The protein expression of total-β-catenin, DAT and HPRT1 as well as the extent of β-catenin phosphorylation in the normal and 6-OHDA-lesioned PD mice. ( D ) TH positive neurons in the substantia nigra tissues examined by immunohistochemistry (upper × 100, lower × 400). ( E – H ) The mRNA expression of Nurr-1 ( E ), Pitx-3 ( F ), Ngn-2 ( G ) and NeuroD1 ( H ) in the substantia nigra tissues examined by RT-qPCR. ( I ) Fluoro-Jade B-stained apoptotic N27 dopaminergic neurons (scale bar = 50 μm). * p

    Journal: Aging (Albany NY)

    Article Title: LncRNA H19 diminishes dopaminergic neuron loss by mediating microRNA-301b-3p in Parkinson’s disease via the HPRT1-mediated Wnt/β-catenin signaling pathway

    doi: 10.18632/aging.102877

    Figure Lengend Snippet: Overexpressed HPRT1 inhibits dopaminergic neuron loss via the Wnt/β-catenin signaling pathway in 6-OHDA-treated N27 dopaminergic neurons and mice. N27 dopaminergic neurons and mice were treated with oe-HPRT1 in the presence of the Wnt/β-catenin blocker XAV-939 or DMSO carrier. ( A ) The protein expression of total-β-catenin, DAT and HPRT1 as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio in the N27 dopaminergic neurons. FOP was designated as background value or as negative control due to its stability. ( C ) The protein expression of total-β-catenin, DAT and HPRT1 as well as the extent of β-catenin phosphorylation in the normal and 6-OHDA-lesioned PD mice. ( D ) TH positive neurons in the substantia nigra tissues examined by immunohistochemistry (upper × 100, lower × 400). ( E – H ) The mRNA expression of Nurr-1 ( E ), Pitx-3 ( F ), Ngn-2 ( G ) and NeuroD1 ( H ) in the substantia nigra tissues examined by RT-qPCR. ( I ) Fluoro-Jade B-stained apoptotic N27 dopaminergic neurons (scale bar = 50 μm). * p

    Article Snippet: ImmunohistochemistryAt day 14 after administration of 6-OHDA, the mice were anesthetized with pentobarbital sodium.

    Techniques: Mouse Assay, Expressing, Western Blot, Activity Assay, Negative Control, Immunohistochemistry, Quantitative RT-PCR, Staining

    Overexpressed H19 activates the Wnt/β-catenin signaling pathway by inhibiting miR-301b-3p. N27 dopaminergic neurons exposed to 6-OHDA were treated with agomir-miR-301b-3p alone or in the presence of oe-H19. ( A ) The protein expression of total-β-catenin and HPRT1 as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio in the N27 dopaminergic neurons. *, p

    Journal: Aging (Albany NY)

    Article Title: LncRNA H19 diminishes dopaminergic neuron loss by mediating microRNA-301b-3p in Parkinson’s disease via the HPRT1-mediated Wnt/β-catenin signaling pathway

    doi: 10.18632/aging.102877

    Figure Lengend Snippet: Overexpressed H19 activates the Wnt/β-catenin signaling pathway by inhibiting miR-301b-3p. N27 dopaminergic neurons exposed to 6-OHDA were treated with agomir-miR-301b-3p alone or in the presence of oe-H19. ( A ) The protein expression of total-β-catenin and HPRT1 as well as the extent of β-catenin phosphorylation in the N27 dopaminergic neurons detected by western blot assay. ( B ) The activity of the Wnt/β-catenin signaling pathway expressed by TOP/FOP ratio in the N27 dopaminergic neurons. *, p

    Article Snippet: ImmunohistochemistryAt day 14 after administration of 6-OHDA, the mice were anesthetized with pentobarbital sodium.

    Techniques: Expressing, Western Blot, Activity Assay

    HPRT1 is poorly expressed in the substantia nigra tissues of 6-OHDA-induced PD mice. ( A ) The expression of HPRT1 in the expression profile of GSE20141 related to PD; ( B ) The expression of HPRT1 in the expression profile of GSE20168 related to PD. ( C ) The protein expression of TH in the substantia nigra tissues of 6-OHDA-induced PD mice measured by western blot analysis. ( D ) Immunohistochemical analysis for the TH positive cells in the substantia nigra tissues of 6-OHDA-induced PD mice (upper × 100, lower × 400); ( E ) Fluoro-Jade B-stained apoptotic neurons (scale bar = 50 μm). ( F ) mRNA expression of HPRT1 in the substantia nigra tissues examined by RT-qPCR; ( G ) Protein expression of HPRT1 in the substantia nigra tissues examined by western blot assay. * p

    Journal: Aging (Albany NY)

    Article Title: LncRNA H19 diminishes dopaminergic neuron loss by mediating microRNA-301b-3p in Parkinson’s disease via the HPRT1-mediated Wnt/β-catenin signaling pathway

    doi: 10.18632/aging.102877

    Figure Lengend Snippet: HPRT1 is poorly expressed in the substantia nigra tissues of 6-OHDA-induced PD mice. ( A ) The expression of HPRT1 in the expression profile of GSE20141 related to PD; ( B ) The expression of HPRT1 in the expression profile of GSE20168 related to PD. ( C ) The protein expression of TH in the substantia nigra tissues of 6-OHDA-induced PD mice measured by western blot analysis. ( D ) Immunohistochemical analysis for the TH positive cells in the substantia nigra tissues of 6-OHDA-induced PD mice (upper × 100, lower × 400); ( E ) Fluoro-Jade B-stained apoptotic neurons (scale bar = 50 μm). ( F ) mRNA expression of HPRT1 in the substantia nigra tissues examined by RT-qPCR; ( G ) Protein expression of HPRT1 in the substantia nigra tissues examined by western blot assay. * p

    Article Snippet: ImmunohistochemistryAt day 14 after administration of 6-OHDA, the mice were anesthetized with pentobarbital sodium.

    Techniques: Mouse Assay, Expressing, Western Blot, Immunohistochemistry, Staining, Quantitative RT-PCR

    H19 inhibits dopaminergic neuron loss by inhibiting miR-301b-3p. 6-OHDA-induced PD model mice were treated with agomir-miR-301b-3p alone or in the presence of oe-H19. ( A ) TH positive neurons in the substantia nigra tissues examined by immunohistochemistry (upper × 100, lower × 400). ( B – E ) The mRNA expression of Nurr-1 ( B ), Pitx-3 ( C ), Ngn-2 ( D ) and NeuroD1 ( E ) in the substantia nigra tissues examined by RT-qPCR. ( F ) Fluoro-Jade B-stained apoptotic neurons in the substantia nigra tissues (scale bar = 50 μm). * p

    Journal: Aging (Albany NY)

    Article Title: LncRNA H19 diminishes dopaminergic neuron loss by mediating microRNA-301b-3p in Parkinson’s disease via the HPRT1-mediated Wnt/β-catenin signaling pathway

    doi: 10.18632/aging.102877

    Figure Lengend Snippet: H19 inhibits dopaminergic neuron loss by inhibiting miR-301b-3p. 6-OHDA-induced PD model mice were treated with agomir-miR-301b-3p alone or in the presence of oe-H19. ( A ) TH positive neurons in the substantia nigra tissues examined by immunohistochemistry (upper × 100, lower × 400). ( B – E ) The mRNA expression of Nurr-1 ( B ), Pitx-3 ( C ), Ngn-2 ( D ) and NeuroD1 ( E ) in the substantia nigra tissues examined by RT-qPCR. ( F ) Fluoro-Jade B-stained apoptotic neurons in the substantia nigra tissues (scale bar = 50 μm). * p

    Article Snippet: ImmunohistochemistryAt day 14 after administration of 6-OHDA, the mice were anesthetized with pentobarbital sodium.

    Techniques: Mouse Assay, Immunohistochemistry, Expressing, Quantitative RT-PCR, Staining

    ECH protected PC1 2 from 6‐OHDA‐induced ERS through promoting seipin ubiquitination and degradation. ( A ) ECH down‐regulated seipin and ERS‐associated protein expression induced by 6‐OHDA. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 3, 40 μg protein each lane). ( B ) Knocking down seipin by siRNA attenuated ERS‐associated protein accumulation induced by 6‐OHDA. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 3, 40 μg protein each lane). SiRNA, 50 nM each group transfected and collected to obtain protein lysis after 48 hrs. ( C ) The relative mRNA levels of seipin were determined using RT‐PCR. The results of RT‐PCR were normalized to GAPDH and expressed as fold change to control. SiRNA of seipin dramatically inhibited mRNA levels of seipin ( P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Echinacoside's nigrostriatal dopaminergic protection against 6‐OHDA‐Induced endoplasmic reticulum stress through reducing the accumulation of Seipin

    doi: 10.1111/jcmm.13285

    Figure Lengend Snippet: ECH protected PC1 2 from 6‐OHDA‐induced ERS through promoting seipin ubiquitination and degradation. ( A ) ECH down‐regulated seipin and ERS‐associated protein expression induced by 6‐OHDA. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 3, 40 μg protein each lane). ( B ) Knocking down seipin by siRNA attenuated ERS‐associated protein accumulation induced by 6‐OHDA. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 3, 40 μg protein each lane). SiRNA, 50 nM each group transfected and collected to obtain protein lysis after 48 hrs. ( C ) The relative mRNA levels of seipin were determined using RT‐PCR. The results of RT‐PCR were normalized to GAPDH and expressed as fold change to control. SiRNA of seipin dramatically inhibited mRNA levels of seipin ( P

    Article Snippet: Briefly, 1 × 109 PC12 cells were harvested after treated with different conditions (6‐OHDA, ECH and 6‐OHDA plus ECH) were harvested and 1 ml complete cell lysis buffer of each group was collected to measure the protein concentration, to prepare Protein A/G Mix magnetic beads (Millipore) and anti‐seipin antibody (5 μg) conjunction according to the instructions and to incubate the prepared beads and 1 mg protein lysis at 2–8°C overnight to capture protein compounds cross‐linked with seipin.

    Techniques: Expressing, Western Blot, Transfection, Lysis, Reverse Transcription Polymerase Chain Reaction

    ECH protected Nigrostriatal neurons from 6‐OHDA‐induced ERS‐associated proteins upregulation and inhibited seipin over accumulation in vivo . ( A ) Protein lysis solution of 5 rats’ striatum in each group pooled together. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 5, 40 μg protein each lane). ( B ) The immunoreactive bands were quantified by densitometry using ImageJ software and normalized to GAPDH. ( n = 3, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Echinacoside's nigrostriatal dopaminergic protection against 6‐OHDA‐Induced endoplasmic reticulum stress through reducing the accumulation of Seipin

    doi: 10.1111/jcmm.13285

    Figure Lengend Snippet: ECH protected Nigrostriatal neurons from 6‐OHDA‐induced ERS‐associated proteins upregulation and inhibited seipin over accumulation in vivo . ( A ) Protein lysis solution of 5 rats’ striatum in each group pooled together. Seipin, GRP94, Bip, ATF‐4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions ( n = 5, 40 μg protein each lane). ( B ) The immunoreactive bands were quantified by densitometry using ImageJ software and normalized to GAPDH. ( n = 3, * P

    Article Snippet: Briefly, 1 × 109 PC12 cells were harvested after treated with different conditions (6‐OHDA, ECH and 6‐OHDA plus ECH) were harvested and 1 ml complete cell lysis buffer of each group was collected to measure the protein concentration, to prepare Protein A/G Mix magnetic beads (Millipore) and anti‐seipin antibody (5 μg) conjunction according to the instructions and to incubate the prepared beads and 1 mg protein lysis at 2–8°C overnight to capture protein compounds cross‐linked with seipin.

    Techniques: In Vivo, Lysis, Expressing, Western Blot, Software

    ECH protected Nigrostriatal neurons from 6‐OHDA‐induced TH downregulation and seipin overexpression in vivo and inhibited ERS‐associated protein Bip expression through seipin in vitro . ( A and B ) Immunofluorescence assay about brain SNpc slices in the model group and 7 mg/kg/day ECH group. Left, untreated side, Right, operation lesion side, Whole, the whole brain(2.5 × objective). The circle showed SNpc area of each side (20 × objective); ( A ) 6‐OHDA caused seipin accumulation and TH expression inhibited in SNpc. ( B ) ECH inhibited seipin and Bip overexpression induced by 6‐OHDA in SNpc. Blue, DAPI; green, Seipin; red, TH; scale bar, 50 μM. ( C and D ) Immunofluorescence assay about PC12 cells treated with different conditions. ( C ) ECH attenuated seipin and Bip expression in cytoplasm. ( D ) Modified seipin expression by siRNA could inhibit Bip overexpression induced by 6‐OHDA. Blue, DAPI; green, Seipin; red, Bip; scale bar, 50 μM.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Echinacoside's nigrostriatal dopaminergic protection against 6‐OHDA‐Induced endoplasmic reticulum stress through reducing the accumulation of Seipin

    doi: 10.1111/jcmm.13285

    Figure Lengend Snippet: ECH protected Nigrostriatal neurons from 6‐OHDA‐induced TH downregulation and seipin overexpression in vivo and inhibited ERS‐associated protein Bip expression through seipin in vitro . ( A and B ) Immunofluorescence assay about brain SNpc slices in the model group and 7 mg/kg/day ECH group. Left, untreated side, Right, operation lesion side, Whole, the whole brain(2.5 × objective). The circle showed SNpc area of each side (20 × objective); ( A ) 6‐OHDA caused seipin accumulation and TH expression inhibited in SNpc. ( B ) ECH inhibited seipin and Bip overexpression induced by 6‐OHDA in SNpc. Blue, DAPI; green, Seipin; red, TH; scale bar, 50 μM. ( C and D ) Immunofluorescence assay about PC12 cells treated with different conditions. ( C ) ECH attenuated seipin and Bip expression in cytoplasm. ( D ) Modified seipin expression by siRNA could inhibit Bip overexpression induced by 6‐OHDA. Blue, DAPI; green, Seipin; red, Bip; scale bar, 50 μM.

    Article Snippet: Briefly, 1 × 109 PC12 cells were harvested after treated with different conditions (6‐OHDA, ECH and 6‐OHDA plus ECH) were harvested and 1 ml complete cell lysis buffer of each group was collected to measure the protein concentration, to prepare Protein A/G Mix magnetic beads (Millipore) and anti‐seipin antibody (5 μg) conjunction according to the instructions and to incubate the prepared beads and 1 mg protein lysis at 2–8°C overnight to capture protein compounds cross‐linked with seipin.

    Techniques: Over Expression, In Vivo, Expressing, In Vitro, Immunofluorescence, Modification

    ECH rescued protein and mRNA expression levels of TH, DAT and decreased α‐synuclein accumulation in 6‐OHDA‐induced rat. ( A ) After intraperitoneally treated with vehicle or 0, 3.5, 7 mg/kg of ECH for 14 days, relative protein levels were measured using Western blotting ( n = 3), GAPDH taken as a reference gene. ( B ) The immunoreactive bands were quantified by densitometry using ImageJ software and normalized to GAPDH ( n = 3, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Echinacoside's nigrostriatal dopaminergic protection against 6‐OHDA‐Induced endoplasmic reticulum stress through reducing the accumulation of Seipin

    doi: 10.1111/jcmm.13285

    Figure Lengend Snippet: ECH rescued protein and mRNA expression levels of TH, DAT and decreased α‐synuclein accumulation in 6‐OHDA‐induced rat. ( A ) After intraperitoneally treated with vehicle or 0, 3.5, 7 mg/kg of ECH for 14 days, relative protein levels were measured using Western blotting ( n = 3), GAPDH taken as a reference gene. ( B ) The immunoreactive bands were quantified by densitometry using ImageJ software and normalized to GAPDH ( n = 3, * P

    Article Snippet: Briefly, 1 × 109 PC12 cells were harvested after treated with different conditions (6‐OHDA, ECH and 6‐OHDA plus ECH) were harvested and 1 ml complete cell lysis buffer of each group was collected to measure the protein concentration, to prepare Protein A/G Mix magnetic beads (Millipore) and anti‐seipin antibody (5 μg) conjunction according to the instructions and to incubate the prepared beads and 1 mg protein lysis at 2–8°C overnight to capture protein compounds cross‐linked with seipin.

    Techniques: Expressing, Western Blot, Software

    Ultrastructural evidence of ECH anti‐ERS (endoplasmic reticulum stress) effects as evaluated by transmission electron microscopy and ECH attenuated the 6‐OHDA‐induced intracellular ROS levels measured by flow cytometry. PC12 cells were pre‐incubated with different concentration of ECH (0, 25, 50, 100 μM) in a serum‐free RPMI‐1640 medium for 1 hr. Then, 6‐OHDA was added to the wells at a final concentration of 100 μM and incubated for another 24 hrs at 37°C. ( A ) 6‐OHDA‐induced ER curled and generated vacuole and ECH rescued ER structural integrity and made it extend. Black arrows, ER; scale bar, 0.5 μm. ( B ) ROS levels were determined using H2DCFH‐DA and measured by flow cytometry. ( C ) Data were calculated as ratio of fluorescence compared with untreated cells. Data are presented as mean ± S.D. values of three independent experiments. n = 3, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Echinacoside's nigrostriatal dopaminergic protection against 6‐OHDA‐Induced endoplasmic reticulum stress through reducing the accumulation of Seipin

    doi: 10.1111/jcmm.13285

    Figure Lengend Snippet: Ultrastructural evidence of ECH anti‐ERS (endoplasmic reticulum stress) effects as evaluated by transmission electron microscopy and ECH attenuated the 6‐OHDA‐induced intracellular ROS levels measured by flow cytometry. PC12 cells were pre‐incubated with different concentration of ECH (0, 25, 50, 100 μM) in a serum‐free RPMI‐1640 medium for 1 hr. Then, 6‐OHDA was added to the wells at a final concentration of 100 μM and incubated for another 24 hrs at 37°C. ( A ) 6‐OHDA‐induced ER curled and generated vacuole and ECH rescued ER structural integrity and made it extend. Black arrows, ER; scale bar, 0.5 μm. ( B ) ROS levels were determined using H2DCFH‐DA and measured by flow cytometry. ( C ) Data were calculated as ratio of fluorescence compared with untreated cells. Data are presented as mean ± S.D. values of three independent experiments. n = 3, * P

    Article Snippet: Briefly, 1 × 109 PC12 cells were harvested after treated with different conditions (6‐OHDA, ECH and 6‐OHDA plus ECH) were harvested and 1 ml complete cell lysis buffer of each group was collected to measure the protein concentration, to prepare Protein A/G Mix magnetic beads (Millipore) and anti‐seipin antibody (5 μg) conjunction according to the instructions and to incubate the prepared beads and 1 mg protein lysis at 2–8°C overnight to capture protein compounds cross‐linked with seipin.

    Techniques: Transmission Assay, Electron Microscopy, Flow Cytometry, Cytometry, Incubation, Concentration Assay, Generated, Fluorescence

    Protective effect of ECH on viability of PC12 cells injured by 6‐OHDA. Cell viability was determined by MTT assay. Data are expressed as percentage of the viability of cells, control taken as 100% viability. ( A ) The chemical structure of echinacoside (ECH). ( B ) Effect of different concentration of ECH on cell viability in PC12 cells (determine the non‐toxic dosages of ECH). PC12 cells were incubated with ECH (12.5–500 μM) for 24 hrs. ( C ) Effect of different concentration of 6‐OHDA on cell viability in PC12 cells (determine the toxic dosages of 6‐OHDA). PC12 cells were treated with various concentrations of 6‐OHDA (0–200 μM) for 24 hrs. ( D ) Effect of ECH on cell viability changes in 6‐OHDA‐induced PC12 cells. PC12 cells were pre‐incubated with different concentration of ECH (0, 25, 50, 100 μM) for 1 hr. Then, 6‐OHDA was added to the wells at a final concentration of 100 μM and incubated for another 24 hrs at 37°C. Data were shown as mean ± S.D., n = 4, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Echinacoside's nigrostriatal dopaminergic protection against 6‐OHDA‐Induced endoplasmic reticulum stress through reducing the accumulation of Seipin

    doi: 10.1111/jcmm.13285

    Figure Lengend Snippet: Protective effect of ECH on viability of PC12 cells injured by 6‐OHDA. Cell viability was determined by MTT assay. Data are expressed as percentage of the viability of cells, control taken as 100% viability. ( A ) The chemical structure of echinacoside (ECH). ( B ) Effect of different concentration of ECH on cell viability in PC12 cells (determine the non‐toxic dosages of ECH). PC12 cells were incubated with ECH (12.5–500 μM) for 24 hrs. ( C ) Effect of different concentration of 6‐OHDA on cell viability in PC12 cells (determine the toxic dosages of 6‐OHDA). PC12 cells were treated with various concentrations of 6‐OHDA (0–200 μM) for 24 hrs. ( D ) Effect of ECH on cell viability changes in 6‐OHDA‐induced PC12 cells. PC12 cells were pre‐incubated with different concentration of ECH (0, 25, 50, 100 μM) for 1 hr. Then, 6‐OHDA was added to the wells at a final concentration of 100 μM and incubated for another 24 hrs at 37°C. Data were shown as mean ± S.D., n = 4, * P

    Article Snippet: Briefly, 1 × 109 PC12 cells were harvested after treated with different conditions (6‐OHDA, ECH and 6‐OHDA plus ECH) were harvested and 1 ml complete cell lysis buffer of each group was collected to measure the protein concentration, to prepare Protein A/G Mix magnetic beads (Millipore) and anti‐seipin antibody (5 μg) conjunction according to the instructions and to incubate the prepared beads and 1 mg protein lysis at 2–8°C overnight to capture protein compounds cross‐linked with seipin.

    Techniques: MTT Assay, Concentration Assay, Incubation

    Nigrostriatal dopaminergic protection of ECH in 6‐OHDA‐induced rat. ( A ) ECH maintained neuronal morphology in striatum: vehicle group, black arrow, angioedema, caused by the surgery; 6‐OHDA group, green arrow, disordered fibre cord, black arrow, white matter oedema; ECH low concentration group, black arrow, white matter oedema; ECH‐high concentration group, no obvious oedema. ( B ) ECH rescued the number of tyrosine hydroxylase(TH)‐immunoreactive DA cells in the substantia nigra (SN) induced by 6‐OHDA damaged. Immunohistochemistry assay of TH taken in 4‐μm paraffin section of the SN. Brown region, TH‐immunoreactive DA cell bodies (20 × objective under white light). ( C ) Relative quantitative counting of TH‐immunoreactive DA cells in each group. The control group taken as 100 per cent compared with other groups ( n = 5, NS, No significant, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Echinacoside's nigrostriatal dopaminergic protection against 6‐OHDA‐Induced endoplasmic reticulum stress through reducing the accumulation of Seipin

    doi: 10.1111/jcmm.13285

    Figure Lengend Snippet: Nigrostriatal dopaminergic protection of ECH in 6‐OHDA‐induced rat. ( A ) ECH maintained neuronal morphology in striatum: vehicle group, black arrow, angioedema, caused by the surgery; 6‐OHDA group, green arrow, disordered fibre cord, black arrow, white matter oedema; ECH low concentration group, black arrow, white matter oedema; ECH‐high concentration group, no obvious oedema. ( B ) ECH rescued the number of tyrosine hydroxylase(TH)‐immunoreactive DA cells in the substantia nigra (SN) induced by 6‐OHDA damaged. Immunohistochemistry assay of TH taken in 4‐μm paraffin section of the SN. Brown region, TH‐immunoreactive DA cell bodies (20 × objective under white light). ( C ) Relative quantitative counting of TH‐immunoreactive DA cells in each group. The control group taken as 100 per cent compared with other groups ( n = 5, NS, No significant, * P

    Article Snippet: Briefly, 1 × 109 PC12 cells were harvested after treated with different conditions (6‐OHDA, ECH and 6‐OHDA plus ECH) were harvested and 1 ml complete cell lysis buffer of each group was collected to measure the protein concentration, to prepare Protein A/G Mix magnetic beads (Millipore) and anti‐seipin antibody (5 μg) conjunction according to the instructions and to incubate the prepared beads and 1 mg protein lysis at 2–8°C overnight to capture protein compounds cross‐linked with seipin.

    Techniques: Concentration Assay, Immunohistochemistry, Paraffin Section

    Using photometry to measure D1R-dependent PKA activation in intact and 6-OHDA lesioned hemi-Parkinsonian rats. Panels a and b show cylinder test results for intact rats and 6-OHDA lesioned rats, respectively. Rats in both groups had been implanted with optic probes and expressed AKAR in the dorsal striatum, in the case of 6-OHDA injected animals this was ipsilateral to the lesion. Y-axes show the percentage of rears in which individual animals used the forelimb that was ipsilateral vs . contralateral to the AAV-injected hemisphere, or used both forelimbs at the same time. Tests in the 6-OHDA lesioned rats occurred 3 weeks following intra-striatal injection of AAV 1 -SynTet-AKAR and optical probe implantation. (c) Representative immunofluorescent images depicting AKAR expression (green) and optic-probe placement in the dorsal striatum in addition to expression of tyrosine hydroxylase (TH, shown in purple) in the striatum, medial forebrain bundle (MFB) and substantia nigra (SN). (d) FRET recordings performed on intact rats expressing AKAR and injected with either saline or the D1R agonist SKF 81297 (0.5 mg/kg s.c.) at t = 10 min (n = 12). (e) FRET recordings performed on 6-OHDA lesioned rats expressing AKAR and injected with either saline or SKF81297 (0.5 mg/kg s.c.) at t = 10 min (n = 9). For all experiments, rats were recorded under both saline and drug conditions in a counterbalanced design. (f) Area under the curve (AUC) analysis of panels d and e. AUC was calculated relative to baseline following drug or saline injection. AUC values for Saline vs . SKF 81297 in each control and lesioned rats were by repeated measures t-tests, * p

    Journal: bioRxiv

    Article Title: Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

    doi: 10.1101/2020.03.12.989103

    Figure Lengend Snippet: Using photometry to measure D1R-dependent PKA activation in intact and 6-OHDA lesioned hemi-Parkinsonian rats. Panels a and b show cylinder test results for intact rats and 6-OHDA lesioned rats, respectively. Rats in both groups had been implanted with optic probes and expressed AKAR in the dorsal striatum, in the case of 6-OHDA injected animals this was ipsilateral to the lesion. Y-axes show the percentage of rears in which individual animals used the forelimb that was ipsilateral vs . contralateral to the AAV-injected hemisphere, or used both forelimbs at the same time. Tests in the 6-OHDA lesioned rats occurred 3 weeks following intra-striatal injection of AAV 1 -SynTet-AKAR and optical probe implantation. (c) Representative immunofluorescent images depicting AKAR expression (green) and optic-probe placement in the dorsal striatum in addition to expression of tyrosine hydroxylase (TH, shown in purple) in the striatum, medial forebrain bundle (MFB) and substantia nigra (SN). (d) FRET recordings performed on intact rats expressing AKAR and injected with either saline or the D1R agonist SKF 81297 (0.5 mg/kg s.c.) at t = 10 min (n = 12). (e) FRET recordings performed on 6-OHDA lesioned rats expressing AKAR and injected with either saline or SKF81297 (0.5 mg/kg s.c.) at t = 10 min (n = 9). For all experiments, rats were recorded under both saline and drug conditions in a counterbalanced design. (f) Area under the curve (AUC) analysis of panels d and e. AUC was calculated relative to baseline following drug or saline injection. AUC values for Saline vs . SKF 81297 in each control and lesioned rats were by repeated measures t-tests, * p

    Article Snippet: Each rat received 2.5 μl of 6-OHDA HBr (7 μg/μL in 0.02% ascorbic acid dissolved in 0.9% NaCl; MilliporeSigma).

    Techniques: Activation Assay, Injection, Expressing

    Immunofluorescent staining for markers of dyskinesia in 6-OHDA lesioned, L-DOPA treated rats. Representative images showing immunolabelling of FosB and serine-10 phosphorylated histone 3 (pSer10-H3) in tissue from 6-OHDA lesioned rats after 2 weeks of daily treatment with L-DOPA/benserazide (6/15 mg/kg s.c.) (n = 5 rats).

    Journal: bioRxiv

    Article Title: Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

    doi: 10.1101/2020.03.12.989103

    Figure Lengend Snippet: Immunofluorescent staining for markers of dyskinesia in 6-OHDA lesioned, L-DOPA treated rats. Representative images showing immunolabelling of FosB and serine-10 phosphorylated histone 3 (pSer10-H3) in tissue from 6-OHDA lesioned rats after 2 weeks of daily treatment with L-DOPA/benserazide (6/15 mg/kg s.c.) (n = 5 rats).

    Article Snippet: Each rat received 2.5 μl of 6-OHDA HBr (7 μg/μL in 0.02% ascorbic acid dissolved in 0.9% NaCl; MilliporeSigma).

    Techniques: Staining

    PKA and ERK1/2 signalling in Parkinsonian rats and following development of L-DOPA-induced dyskinesia. (a) Timeline of surgeries, drug treatments and testing for L-DOPA-induced dyskinesia (LID) experiments. (b) PKA biosensor responses to saline or SKF 81297 (0.5 mg/kg s.c) in drug naive 6-OHDA lesioned rats. The left panel shows time-course of FRET responses, while the right panel shows the integrated area-under the curve (AUC) for the post-injection period. All animals were tested under both saline and drug conditions, as indicated by the connecting lines on the AUC plot (n = 14 rats). (c) ERK1/2 biosensor responses to saline or SKF 81297 in drug naive 6-OHDA lesioned rats. The left panel shows the time-course of FRET responses, while the right panel shows the integrated area-under the curve (AUC) for the post-injection period. All animals were tested under both saline and drug conditions, as indicated by the connecting lines on the AUC plot (n = 8 rats). (d) 6-OHDA lesioned rats develop L-DOPA-induced dyskinesia following 2 weeks of daily treatment with L-DOPA/benserazide (6/15 mg/kg s.c.). Dyskinesia was measured as an AIM score every 10 min (red line), while PKA activation was simultaneously recorded at 2 min intervals (blue line) (n = 14 rats). (e) PKA biosensor responses to saline or SKF 81297 in L-DOPA primed, dyskinetic rats, either 24 or 48 hours after the last L-DOPA treatment. The left panel shows time-course of FRET responses, while the right panel shows the integrated area-under the curve (AUC) for the post-injection period (n = 5, 14). (f) ERK1/2 biosensor responses to saline or SKF 81297 in L-DOPA primed, dyskinetic rats, either 24 or 48 hours after the last L-DOPA treatment. The left panel shows time-course of FRET responses, while the right panel shows the integrated area-under the curve (AUC) for the post-injection period (n = 6-8 rats). (g) Representative immunofluorescent images depicting phospho-ERK1/2 (pERK) immunolabelling in L-DOPA primed, dyskinetic rats challenged with either saline (n = 3) or SKF 81297 (n = 4), 24 hours following the last L-DOPA treatment. Data in all time-course plots are mean ± SEM. Statistical analysis in panels b and c was performed by paired t-test. Statistical analysis in panels e and f was performed by paired t-test with Bonferroni correction, * p

    Journal: bioRxiv

    Article Title: Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

    doi: 10.1101/2020.03.12.989103

    Figure Lengend Snippet: PKA and ERK1/2 signalling in Parkinsonian rats and following development of L-DOPA-induced dyskinesia. (a) Timeline of surgeries, drug treatments and testing for L-DOPA-induced dyskinesia (LID) experiments. (b) PKA biosensor responses to saline or SKF 81297 (0.5 mg/kg s.c) in drug naive 6-OHDA lesioned rats. The left panel shows time-course of FRET responses, while the right panel shows the integrated area-under the curve (AUC) for the post-injection period. All animals were tested under both saline and drug conditions, as indicated by the connecting lines on the AUC plot (n = 14 rats). (c) ERK1/2 biosensor responses to saline or SKF 81297 in drug naive 6-OHDA lesioned rats. The left panel shows the time-course of FRET responses, while the right panel shows the integrated area-under the curve (AUC) for the post-injection period. All animals were tested under both saline and drug conditions, as indicated by the connecting lines on the AUC plot (n = 8 rats). (d) 6-OHDA lesioned rats develop L-DOPA-induced dyskinesia following 2 weeks of daily treatment with L-DOPA/benserazide (6/15 mg/kg s.c.). Dyskinesia was measured as an AIM score every 10 min (red line), while PKA activation was simultaneously recorded at 2 min intervals (blue line) (n = 14 rats). (e) PKA biosensor responses to saline or SKF 81297 in L-DOPA primed, dyskinetic rats, either 24 or 48 hours after the last L-DOPA treatment. The left panel shows time-course of FRET responses, while the right panel shows the integrated area-under the curve (AUC) for the post-injection period (n = 5, 14). (f) ERK1/2 biosensor responses to saline or SKF 81297 in L-DOPA primed, dyskinetic rats, either 24 or 48 hours after the last L-DOPA treatment. The left panel shows time-course of FRET responses, while the right panel shows the integrated area-under the curve (AUC) for the post-injection period (n = 6-8 rats). (g) Representative immunofluorescent images depicting phospho-ERK1/2 (pERK) immunolabelling in L-DOPA primed, dyskinetic rats challenged with either saline (n = 3) or SKF 81297 (n = 4), 24 hours following the last L-DOPA treatment. Data in all time-course plots are mean ± SEM. Statistical analysis in panels b and c was performed by paired t-test. Statistical analysis in panels e and f was performed by paired t-test with Bonferroni correction, * p

    Article Snippet: Each rat received 2.5 μl of 6-OHDA HBr (7 μg/μL in 0.02% ascorbic acid dissolved in 0.9% NaCl; MilliporeSigma).

    Techniques: Injection, Activation Assay

    D1R agonist-induced PKA and ERK1/2 signalling is significantly attenuated following chronic L-DOPA, while dyskinesia is significantly enhanced PKA and ERK1/2 biosensor response profiles (%ΔF/F) and dyskinesia scores (AIMS) in drug naïve, 6-OHDA lesioned rats and L-DOPA primed, dyskinetic rats treated with the D1R agonist SKF 81297 (0.5 mg/kg s.c.) (a) PKA biosensor response profiles and dyskinesia scores in drug-naïve, 6-OHDA lesioned rats treated with SKF 81297 (0.5 mg/kg s.c.) (n = 14 rats) (b) PKA biosensor response profiles and dyskinesia scores in L-DOPA primed, dyskinetic rats treated with SKF 81297 (n = 14 rats). (c) ERK1/2 biosensor response profiles and dyskinesia scores in drug naïve, 6-OHDA lesioned rats treated with SKF 81297 (n = 9 rats) (d) ERK1/2 response profile and dyskinesia score in L-DOPA primed, dyskinetic rats treated with 0.5mg/kg SKF81297 (n = 8 rats). (e) Integrated area-under the curve (AUC) for the post-injection period comparing PKA activation by saline versus SKF 81297 on Day 0 (pre-priming) and Day 15 (post-priming). (f) Comparison of peak dyskinesia (measured at 40 min post-injection) on Day 0 versus Day 15 in AKAR rats. (g) Integrated area-under the curve (AUC) for the post-injection period comparing ERK1/2 activation by saline versus SKF 81297 on Day 0 (pre-priming) and Day 15 (post-priming). (h) Comparison of peak dyskinesia (measured at 40 min post-injection) on Day 0 versus Day 15 in EKAR rats. All data is presented as mean ± SEM. Statistical analysis of photometry data before and after L-DOPA priming (e, g) was performed using paired t-test with Bonferroni correction. For EKAR data, one rat was excluded from the repeated measures ANOVA due to a missing value. Comparison of dyskinesia (f, h) was performed by paired t-test, * p

    Journal: bioRxiv

    Article Title: Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

    doi: 10.1101/2020.03.12.989103

    Figure Lengend Snippet: D1R agonist-induced PKA and ERK1/2 signalling is significantly attenuated following chronic L-DOPA, while dyskinesia is significantly enhanced PKA and ERK1/2 biosensor response profiles (%ΔF/F) and dyskinesia scores (AIMS) in drug naïve, 6-OHDA lesioned rats and L-DOPA primed, dyskinetic rats treated with the D1R agonist SKF 81297 (0.5 mg/kg s.c.) (a) PKA biosensor response profiles and dyskinesia scores in drug-naïve, 6-OHDA lesioned rats treated with SKF 81297 (0.5 mg/kg s.c.) (n = 14 rats) (b) PKA biosensor response profiles and dyskinesia scores in L-DOPA primed, dyskinetic rats treated with SKF 81297 (n = 14 rats). (c) ERK1/2 biosensor response profiles and dyskinesia scores in drug naïve, 6-OHDA lesioned rats treated with SKF 81297 (n = 9 rats) (d) ERK1/2 response profile and dyskinesia score in L-DOPA primed, dyskinetic rats treated with 0.5mg/kg SKF81297 (n = 8 rats). (e) Integrated area-under the curve (AUC) for the post-injection period comparing PKA activation by saline versus SKF 81297 on Day 0 (pre-priming) and Day 15 (post-priming). (f) Comparison of peak dyskinesia (measured at 40 min post-injection) on Day 0 versus Day 15 in AKAR rats. (g) Integrated area-under the curve (AUC) for the post-injection period comparing ERK1/2 activation by saline versus SKF 81297 on Day 0 (pre-priming) and Day 15 (post-priming). (h) Comparison of peak dyskinesia (measured at 40 min post-injection) on Day 0 versus Day 15 in EKAR rats. All data is presented as mean ± SEM. Statistical analysis of photometry data before and after L-DOPA priming (e, g) was performed using paired t-test with Bonferroni correction. For EKAR data, one rat was excluded from the repeated measures ANOVA due to a missing value. Comparison of dyskinesia (f, h) was performed by paired t-test, * p

    Article Snippet: Each rat received 2.5 μl of 6-OHDA HBr (7 μg/μL in 0.02% ascorbic acid dissolved in 0.9% NaCl; MilliporeSigma).

    Techniques: Injection, Activation Assay