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Enhanced prostacyclin synthesis by adenoviral gene transfer reduced glial activation and ameliorated dopaminergic dysfunction in hemiparkinsonian rats.

Tsai MJ, Weng CF, Yu NC, Liou DY, Kuo FS, Huang MC, Huang WC, Tam K, Shyue SK, Cheng H - Oxid Med Cell Longev (2013)

Bottom Line: Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation.Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats.Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway.

View Article: PubMed Central - PubMed

Affiliation: Neural Regeneration Laboratory, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei 11221, Taiwan.

ABSTRACT
Prostacyclin (PGI2), a potent vasodilator and platelet antiaggregatory eicosanoid, is cytoprotective in cerebral circulation. It is synthesized from arachidonic acid (AA) by the sequential action of cyclooxygenase- (COX-) 1 or 2 and prostacyclin synthase (PGIS). Because prostacyclin is unstable in vivo, PGI2 analogs have been developed and demonstrated to protect against brain ischemia. This work attempts to selectively augment PGI2 synthesis in mixed glial culture or in a model of Parkinson's disease (PD) by direct adenoviral gene transfer of prostacyclin biosynthetic enzymes and examines whether it confers protection in cultures or in vivo. Confluent mixed glial cultures actively metabolized exogenous AA into PGE2 and PGD2. These PGs were largely NS398 sensitive and considered as COX-2 products. Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation. Furthermore, PGIS overexpression significantly reduced LPS stimulation in cultures. In vivo, adenoviral gene transfer of bicistronic COX-1/PGIS to substantia nigra protected 6-OHDA- induced dopamine depletion and ameliorated behavioral deficits. Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats. Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway.

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Infective tropism of adenovirus encoding GFP (AdGFP), which was injected into the substantia nigra (SN) 3 days ago. Representative micrographs of double labeling staining of coronal sections in the injection site. (a)~(d) Photos (magnification ×200) of each double staining; green color: GFP immunoreactivity (IR); red color: TH-IR (panel a), nestin-IR (panel b), GSLI-IB4-IR (panel c) and GFAP (panels d). Arrows or arrow heads in the figures indicate double-IR. AdGFP could transduce dopaminergic (TH-IR) neurons and GSLI-IB4-IR microglia in the SN.
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fig5: Infective tropism of adenovirus encoding GFP (AdGFP), which was injected into the substantia nigra (SN) 3 days ago. Representative micrographs of double labeling staining of coronal sections in the injection site. (a)~(d) Photos (magnification ×200) of each double staining; green color: GFP immunoreactivity (IR); red color: TH-IR (panel a), nestin-IR (panel b), GSLI-IB4-IR (panel c) and GFAP (panels d). Arrows or arrow heads in the figures indicate double-IR. AdGFP could transduce dopaminergic (TH-IR) neurons and GSLI-IB4-IR microglia in the SN.

Mentions: First, we examined the infective tropism of AdGFP in mesencephalic neuron/glial cultures which were enriched with DA neurons. AdGFP (~106 pfu/well each) was added to cultured cells in serum or serum-free medium. As shown in Figure 4, AdGFP predominantly transduced nonneuronal cells. In serum-free condition, the AdGFP infective cells were 19.6 ± 2.9% astroglial (GFAP-positive) cells, 24.4 ± 6.1% microglia (ED1-positive) cells, and 43.9 ± 4% NG2-positive cells. No βIII tubulin-positive neuron or TH-positive DA neurons showed GFP immunoreactivity. Furthermore, the AdGFP infective efficiency in serum-containing medium (10% FCS) was reduced compared to that in serum-free condition. Using same titer of AdGFP to neuron/glial culture, GFP-positive cells in cultures maintained in serum-free and serum-containing conditions were 31.83 ± 3.12 and 19.89 ± 2.19 cells/mm2, respectively. We further performed double-labelled staining for AdGFP-infected coronal sections containing SN. After injection of AdGFP into the normal SN, the expression of GFP was observed along the site of injection. Figure 5 shows the representative micrographs of double-labelled immunostaining results. Some GFP-positive cells in the SN were also positively stained for TH, which denote dopaminergic neurons (Figure 5(a), arrow). Some GSLI-IB4-positive microglia (in Figure 5(b), arrow head) seemed to be immunoreactive to GFP. By contrast, almost no double-labelled staining cells were found in GFP with GFAP or nestin immunoreactivity (Figures 5(c) and 5(d)). These results showed that transgene expression could be achieved in neurons or microglia after injection of AdGFP into the SN in vivo. The results of AdGFP tropism in cultures and in SN were not consistent. We also infected AdGFP to the striatum and examined the infective tropism (shown in Supplementary Figure 2). Consistent with the results observed in AdGFP-infected SN in vivo, TH-positive, GFAP-positive, ED1-positive and nestin-positive cells were found to be double labelled with GFP in rat striatum.


Enhanced prostacyclin synthesis by adenoviral gene transfer reduced glial activation and ameliorated dopaminergic dysfunction in hemiparkinsonian rats.

Tsai MJ, Weng CF, Yu NC, Liou DY, Kuo FS, Huang MC, Huang WC, Tam K, Shyue SK, Cheng H - Oxid Med Cell Longev (2013)

Infective tropism of adenovirus encoding GFP (AdGFP), which was injected into the substantia nigra (SN) 3 days ago. Representative micrographs of double labeling staining of coronal sections in the injection site. (a)~(d) Photos (magnification ×200) of each double staining; green color: GFP immunoreactivity (IR); red color: TH-IR (panel a), nestin-IR (panel b), GSLI-IB4-IR (panel c) and GFAP (panels d). Arrows or arrow heads in the figures indicate double-IR. AdGFP could transduce dopaminergic (TH-IR) neurons and GSLI-IB4-IR microglia in the SN.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649752&req=5

fig5: Infective tropism of adenovirus encoding GFP (AdGFP), which was injected into the substantia nigra (SN) 3 days ago. Representative micrographs of double labeling staining of coronal sections in the injection site. (a)~(d) Photos (magnification ×200) of each double staining; green color: GFP immunoreactivity (IR); red color: TH-IR (panel a), nestin-IR (panel b), GSLI-IB4-IR (panel c) and GFAP (panels d). Arrows or arrow heads in the figures indicate double-IR. AdGFP could transduce dopaminergic (TH-IR) neurons and GSLI-IB4-IR microglia in the SN.
Mentions: First, we examined the infective tropism of AdGFP in mesencephalic neuron/glial cultures which were enriched with DA neurons. AdGFP (~106 pfu/well each) was added to cultured cells in serum or serum-free medium. As shown in Figure 4, AdGFP predominantly transduced nonneuronal cells. In serum-free condition, the AdGFP infective cells were 19.6 ± 2.9% astroglial (GFAP-positive) cells, 24.4 ± 6.1% microglia (ED1-positive) cells, and 43.9 ± 4% NG2-positive cells. No βIII tubulin-positive neuron or TH-positive DA neurons showed GFP immunoreactivity. Furthermore, the AdGFP infective efficiency in serum-containing medium (10% FCS) was reduced compared to that in serum-free condition. Using same titer of AdGFP to neuron/glial culture, GFP-positive cells in cultures maintained in serum-free and serum-containing conditions were 31.83 ± 3.12 and 19.89 ± 2.19 cells/mm2, respectively. We further performed double-labelled staining for AdGFP-infected coronal sections containing SN. After injection of AdGFP into the normal SN, the expression of GFP was observed along the site of injection. Figure 5 shows the representative micrographs of double-labelled immunostaining results. Some GFP-positive cells in the SN were also positively stained for TH, which denote dopaminergic neurons (Figure 5(a), arrow). Some GSLI-IB4-positive microglia (in Figure 5(b), arrow head) seemed to be immunoreactive to GFP. By contrast, almost no double-labelled staining cells were found in GFP with GFAP or nestin immunoreactivity (Figures 5(c) and 5(d)). These results showed that transgene expression could be achieved in neurons or microglia after injection of AdGFP into the SN in vivo. The results of AdGFP tropism in cultures and in SN were not consistent. We also infected AdGFP to the striatum and examined the infective tropism (shown in Supplementary Figure 2). Consistent with the results observed in AdGFP-infected SN in vivo, TH-positive, GFAP-positive, ED1-positive and nestin-positive cells were found to be double labelled with GFP in rat striatum.

Bottom Line: Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation.Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats.Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway.

View Article: PubMed Central - PubMed

Affiliation: Neural Regeneration Laboratory, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei 11221, Taiwan.

ABSTRACT
Prostacyclin (PGI2), a potent vasodilator and platelet antiaggregatory eicosanoid, is cytoprotective in cerebral circulation. It is synthesized from arachidonic acid (AA) by the sequential action of cyclooxygenase- (COX-) 1 or 2 and prostacyclin synthase (PGIS). Because prostacyclin is unstable in vivo, PGI2 analogs have been developed and demonstrated to protect against brain ischemia. This work attempts to selectively augment PGI2 synthesis in mixed glial culture or in a model of Parkinson's disease (PD) by direct adenoviral gene transfer of prostacyclin biosynthetic enzymes and examines whether it confers protection in cultures or in vivo. Confluent mixed glial cultures actively metabolized exogenous AA into PGE2 and PGD2. These PGs were largely NS398 sensitive and considered as COX-2 products. Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation. Furthermore, PGIS overexpression significantly reduced LPS stimulation in cultures. In vivo, adenoviral gene transfer of bicistronic COX-1/PGIS to substantia nigra protected 6-OHDA- induced dopamine depletion and ameliorated behavioral deficits. Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats. Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway.

Show MeSH
Related in: MedlinePlus