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Activation of GSK-3β and caspase-3 occurs in Nigral dopamine neurons during the development of apoptosis activated by a striatal injection of 6-hydroxydopamine.

Hernandez-Baltazar D, Mendoza-Garrido ME, Martinez-Fong D - PLoS ONE (2013)

Bottom Line: The disappearance of TH(+) cells was associated with a decrease in NeuN and β-III tubulin immunoreactivity and an increase in Apostain, cleaved caspase-3, and GSK-3β pY216 in the SNc.Increased levels of caspase-3 immunoreactivity in TH(+) cells were detected from days 1 to 15, and the levels then decreased to day 30 postlesion.Our results suggest that caspase-3 and GSK-3β pY216 activation might participate in the DA cell death and that the active caspase-3 might also participate in the neuroinflammation caused by the striatal 6-OHDA injection.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, DF, México.

ABSTRACT
The 6-Hydroxydopamine (6-OHDA) rat model of Parkinson's disease is essential for a better understanding of the pathological processes underlying the human disease and for the evaluation of promising therapeutic interventions. This work evaluated whether a single striatal injection of 6-OHDA causes progressive apoptosis of dopamine (DA) neurons and activation of glycogen synthase kinase 3β (GSK-3β) and caspase-3 in the substantia nigra compacta (SNc). The loss of DA neurons was shown by three neuron markers; tyrosine hydroxylase (TH), NeuN, and β-III tubulin. Apoptosis activation was determined using Apostain and immunostaining against cleaved caspase-3 and GSK-3β pY216. We also explored the possibility that cleaved caspase-3 is produced by microglia and astrocytes. Our results showed that the 6-OHDA caused loss of nigral TH(+) cells, progressing mainly in rostrocaudal and lateromedial directions. In the neostriatum, a severe loss of TH(+) terminals occurred from day 3 after lesion. The disappearance of TH(+) cells was associated with a decrease in NeuN and β-III tubulin immunoreactivity and an increase in Apostain, cleaved caspase-3, and GSK-3β pY216 in the SNc. Apostain immunoreactivity was observed from days 3 to 21 postlesion. Increased levels of caspase-3 immunoreactivity in TH(+) cells were detected from days 1 to 15, and the levels then decreased to day 30 postlesion. The cleaved caspase-3 also collocated with microglia and astrocytes indicating its participation in glial activation. Our results suggest that caspase-3 and GSK-3β pY216 activation might participate in the DA cell death and that the active caspase-3 might also participate in the neuroinflammation caused by the striatal 6-OHDA injection.

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Progressive loss of TH(+) cells in the SNc after a 6-OHDA injection into the neostriatum.The cell counting was done on 10 to 11 coronal slices (35 µm) per rat (n = 3 per group). In panel A, representative micrographs of TH-immunostained slices taken from the rostral, medial, and caudal mesencephalon. In graphs B and C, cell counting in rostrocaudal direction and lateromedial direction respectively. In D, total counts of residual TH(+) cells. In E and F, representative micrograph showing TH immunostaining and cell counting in the VTA. The primary antibody was a mouse monoclonal anti-TH clone TH-2 and the secondary antibody was a horse biotinylated anti-mouse IgG (H+L). All values are expressed as the mean ± SEM *P<0.05, **P<0.001 when compared with the intact group or between groups; one-way ANOVA test and Newman-Keuls post-hoc test. NS = not significant. The scale bars = 1 mm in the first row (control) are common for all micrographs.
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pone-0070951-g001: Progressive loss of TH(+) cells in the SNc after a 6-OHDA injection into the neostriatum.The cell counting was done on 10 to 11 coronal slices (35 µm) per rat (n = 3 per group). In panel A, representative micrographs of TH-immunostained slices taken from the rostral, medial, and caudal mesencephalon. In graphs B and C, cell counting in rostrocaudal direction and lateromedial direction respectively. In D, total counts of residual TH(+) cells. In E and F, representative micrograph showing TH immunostaining and cell counting in the VTA. The primary antibody was a mouse monoclonal anti-TH clone TH-2 and the secondary antibody was a horse biotinylated anti-mouse IgG (H+L). All values are expressed as the mean ± SEM *P<0.05, **P<0.001 when compared with the intact group or between groups; one-way ANOVA test and Newman-Keuls post-hoc test. NS = not significant. The scale bars = 1 mm in the first row (control) are common for all micrographs.

Mentions: A single injection of 6-OHDA into the neostriatum mainly decreased the number of TH-immunoreactive cells in the SNc (Fig. 1A–D) and in a lesser proportion in the VTA (Fig. 1E–F). A 25% significant decrease in TH(+) cells was measured as early as three days postlesion in the three regions of the SNc studied (rostral, medial, and caudal) as compared with those of the intact control side (Fig. 1A–D). The loss of TH(+) cells progressively continued up to 30 days postlesion and was 90% as compared with the intact control (Fig. 1A,D). The loss of TH(+) cells progressed mainly in the rostrocaudal direction (Fig. 1B) and in the lateromedial direction (Fig. 1C). When the comparison between regions (rostral, medial, and caudal, or lateral, medial, and interior) was made, the counts of TH(+) cells showed a statistically significant difference from 3 to 15 days postlesion (Fig. 1A–C). The population of TH(+) cells in the VTA decreased 20% at day 7 and remained at that same percentage to the end of the study (Fig. 1E,F). The decrease in TH immunoreactivity caused by 6-OHDA in the neostriatum appeared earlier than in the substantia nigra (at day 3 postlesion) because of the close proximity with the site of the neurotoxin injection (Fig. S1).


Activation of GSK-3β and caspase-3 occurs in Nigral dopamine neurons during the development of apoptosis activated by a striatal injection of 6-hydroxydopamine.

Hernandez-Baltazar D, Mendoza-Garrido ME, Martinez-Fong D - PLoS ONE (2013)

Progressive loss of TH(+) cells in the SNc after a 6-OHDA injection into the neostriatum.The cell counting was done on 10 to 11 coronal slices (35 µm) per rat (n = 3 per group). In panel A, representative micrographs of TH-immunostained slices taken from the rostral, medial, and caudal mesencephalon. In graphs B and C, cell counting in rostrocaudal direction and lateromedial direction respectively. In D, total counts of residual TH(+) cells. In E and F, representative micrograph showing TH immunostaining and cell counting in the VTA. The primary antibody was a mouse monoclonal anti-TH clone TH-2 and the secondary antibody was a horse biotinylated anti-mouse IgG (H+L). All values are expressed as the mean ± SEM *P<0.05, **P<0.001 when compared with the intact group or between groups; one-way ANOVA test and Newman-Keuls post-hoc test. NS = not significant. The scale bars = 1 mm in the first row (control) are common for all micrographs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3733721&req=5

pone-0070951-g001: Progressive loss of TH(+) cells in the SNc after a 6-OHDA injection into the neostriatum.The cell counting was done on 10 to 11 coronal slices (35 µm) per rat (n = 3 per group). In panel A, representative micrographs of TH-immunostained slices taken from the rostral, medial, and caudal mesencephalon. In graphs B and C, cell counting in rostrocaudal direction and lateromedial direction respectively. In D, total counts of residual TH(+) cells. In E and F, representative micrograph showing TH immunostaining and cell counting in the VTA. The primary antibody was a mouse monoclonal anti-TH clone TH-2 and the secondary antibody was a horse biotinylated anti-mouse IgG (H+L). All values are expressed as the mean ± SEM *P<0.05, **P<0.001 when compared with the intact group or between groups; one-way ANOVA test and Newman-Keuls post-hoc test. NS = not significant. The scale bars = 1 mm in the first row (control) are common for all micrographs.
Mentions: A single injection of 6-OHDA into the neostriatum mainly decreased the number of TH-immunoreactive cells in the SNc (Fig. 1A–D) and in a lesser proportion in the VTA (Fig. 1E–F). A 25% significant decrease in TH(+) cells was measured as early as three days postlesion in the three regions of the SNc studied (rostral, medial, and caudal) as compared with those of the intact control side (Fig. 1A–D). The loss of TH(+) cells progressively continued up to 30 days postlesion and was 90% as compared with the intact control (Fig. 1A,D). The loss of TH(+) cells progressed mainly in the rostrocaudal direction (Fig. 1B) and in the lateromedial direction (Fig. 1C). When the comparison between regions (rostral, medial, and caudal, or lateral, medial, and interior) was made, the counts of TH(+) cells showed a statistically significant difference from 3 to 15 days postlesion (Fig. 1A–C). The population of TH(+) cells in the VTA decreased 20% at day 7 and remained at that same percentage to the end of the study (Fig. 1E,F). The decrease in TH immunoreactivity caused by 6-OHDA in the neostriatum appeared earlier than in the substantia nigra (at day 3 postlesion) because of the close proximity with the site of the neurotoxin injection (Fig. S1).

Bottom Line: The disappearance of TH(+) cells was associated with a decrease in NeuN and β-III tubulin immunoreactivity and an increase in Apostain, cleaved caspase-3, and GSK-3β pY216 in the SNc.Increased levels of caspase-3 immunoreactivity in TH(+) cells were detected from days 1 to 15, and the levels then decreased to day 30 postlesion.Our results suggest that caspase-3 and GSK-3β pY216 activation might participate in the DA cell death and that the active caspase-3 might also participate in the neuroinflammation caused by the striatal 6-OHDA injection.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, DF, México.

ABSTRACT
The 6-Hydroxydopamine (6-OHDA) rat model of Parkinson's disease is essential for a better understanding of the pathological processes underlying the human disease and for the evaluation of promising therapeutic interventions. This work evaluated whether a single striatal injection of 6-OHDA causes progressive apoptosis of dopamine (DA) neurons and activation of glycogen synthase kinase 3β (GSK-3β) and caspase-3 in the substantia nigra compacta (SNc). The loss of DA neurons was shown by three neuron markers; tyrosine hydroxylase (TH), NeuN, and β-III tubulin. Apoptosis activation was determined using Apostain and immunostaining against cleaved caspase-3 and GSK-3β pY216. We also explored the possibility that cleaved caspase-3 is produced by microglia and astrocytes. Our results showed that the 6-OHDA caused loss of nigral TH(+) cells, progressing mainly in rostrocaudal and lateromedial directions. In the neostriatum, a severe loss of TH(+) terminals occurred from day 3 after lesion. The disappearance of TH(+) cells was associated with a decrease in NeuN and β-III tubulin immunoreactivity and an increase in Apostain, cleaved caspase-3, and GSK-3β pY216 in the SNc. Apostain immunoreactivity was observed from days 3 to 21 postlesion. Increased levels of caspase-3 immunoreactivity in TH(+) cells were detected from days 1 to 15, and the levels then decreased to day 30 postlesion. The cleaved caspase-3 also collocated with microglia and astrocytes indicating its participation in glial activation. Our results suggest that caspase-3 and GSK-3β pY216 activation might participate in the DA cell death and that the active caspase-3 might also participate in the neuroinflammation caused by the striatal 6-OHDA injection.

Show MeSH
Related in: MedlinePlus