Role of MAPK in neuroprotection by cart in ischemic stroke

Yun Xu, Jia Jia, Zichun Hua, Patricia Hurn, Nabil Alkayed

Research output: Contribution to journalArticle

Abstract

BACKGROUND AND SIGNIFICANCE: Our previous work indicated that CART (cocaine and amphetamine regulated transcript) plays an important role in estrogen-mediated neuroprotection in ischemic stroke (Xu Y et al. 2006). Specifically, we previously demonstrated that CART protects primary cultured cortical neurons from cell death induced by oxygen-glucose deprivation (OGD) and decreases infarct size after ischemic stroke induced in mice by middle cerebral artery occlusion (MCAO). The aims of this study are: 1) to further investigate the neuroprotective role of CART in vitro and in vivo using small interfering RNA (siRNA) to downregulate endogenous CART, and 2) to explore a potential mechanism of neuroprotection by CART. CART and its mechanism of action may serve as a novel therapeutic strategy for neuroprotection in ischemic stroke. METHODS: Five double-stranded RNA fragments corresponding to selected mouse CART mRNA sites were produced using plasmid siRNA expression vector under control of U6 promoter. The vector also co-expresses the coral GFP marker (cGFP) under CMV promoter, which is used to track transfection efficiency. Cortical neurons and HEK293T cells were transfected with CART siRNA or control vector, and interference efficiency was assayed by RT-PCR and ELISA. HEK293 cells were also co-transfected with CART cDNA, since they do not express CART. Finally, CART or vehicle, CART siRNA or control vector in vehicle (cationic polymer polyethylenimine, PEI) were injected into mouse lateral ventricles in vivo 24 hours prior to MCAO. Brains were removed, stained with 2,3,5- triphenyltetrazolium chloride (TTC) for infarct size measurement. Neuronal cell death in vitro was induced by OGD, and assayed by propidium iodide (PI)/Calcein staining, and survival assayed by MTT. The phosphorylation of ERK1/2 was measured by western blot with anti-ERK anti-p(44/42)ERK antibodies. RESULTS: CART expression was effectively suppressed by siCART. Selected siRNA reduced CART RNA and protein levels in vitro by approximately 80% and 90%, respectively. CART administration reduced infarct size at 24 hrs after MCAO from 39·6% to 22·4% (n=6 per group, p

Original languageEnglish (US)
JournalJournal of Cerebral Blood Flow and Metabolism
Volume27
Issue numberSUPPL. 1
StatePublished - Nov 13 2007
Externally publishedYes

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Amphetamine
Cocaine
Stroke
Small Interfering RNA
Middle Cerebral Artery Infarction
Neuroprotection
Cell Death
Oxygen
Neurons
Polyethyleneimine
Glucose
Anthozoa
Double-Stranded RNA
Propidium
Lateral Ventricles
HEK293 Cells
Transfection
Polymers
Estrogens
Plasmids

ASJC Scopus subject areas

  • Endocrinology
  • Neuroscience(all)
  • Endocrinology, Diabetes and Metabolism

Cite this

Role of MAPK in neuroprotection by cart in ischemic stroke. / Xu, Yun; Jia, Jia; Hua, Zichun; Hurn, Patricia; Alkayed, Nabil.

In: Journal of Cerebral Blood Flow and Metabolism, Vol. 27, No. SUPPL. 1, 13.11.2007.

Research output: Contribution to journalArticle

Xu, Yun ; Jia, Jia ; Hua, Zichun ; Hurn, Patricia ; Alkayed, Nabil. / Role of MAPK in neuroprotection by cart in ischemic stroke. In: Journal of Cerebral Blood Flow and Metabolism. 2007 ; Vol. 27, No. SUPPL. 1.
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abstract = "BACKGROUND AND SIGNIFICANCE: Our previous work indicated that CART (cocaine and amphetamine regulated transcript) plays an important role in estrogen-mediated neuroprotection in ischemic stroke (Xu Y et al. 2006). Specifically, we previously demonstrated that CART protects primary cultured cortical neurons from cell death induced by oxygen-glucose deprivation (OGD) and decreases infarct size after ischemic stroke induced in mice by middle cerebral artery occlusion (MCAO). The aims of this study are: 1) to further investigate the neuroprotective role of CART in vitro and in vivo using small interfering RNA (siRNA) to downregulate endogenous CART, and 2) to explore a potential mechanism of neuroprotection by CART. CART and its mechanism of action may serve as a novel therapeutic strategy for neuroprotection in ischemic stroke. METHODS: Five double-stranded RNA fragments corresponding to selected mouse CART mRNA sites were produced using plasmid siRNA expression vector under control of U6 promoter. The vector also co-expresses the coral GFP marker (cGFP) under CMV promoter, which is used to track transfection efficiency. Cortical neurons and HEK293T cells were transfected with CART siRNA or control vector, and interference efficiency was assayed by RT-PCR and ELISA. HEK293 cells were also co-transfected with CART cDNA, since they do not express CART. Finally, CART or vehicle, CART siRNA or control vector in vehicle (cationic polymer polyethylenimine, PEI) were injected into mouse lateral ventricles in vivo 24 hours prior to MCAO. Brains were removed, stained with 2,3,5- triphenyltetrazolium chloride (TTC) for infarct size measurement. Neuronal cell death in vitro was induced by OGD, and assayed by propidium iodide (PI)/Calcein staining, and survival assayed by MTT. The phosphorylation of ERK1/2 was measured by western blot with anti-ERK anti-p(44/42)ERK antibodies. RESULTS: CART expression was effectively suppressed by siCART. Selected siRNA reduced CART RNA and protein levels in vitro by approximately 80{\%} and 90{\%}, respectively. CART administration reduced infarct size at 24 hrs after MCAO from 39·6{\%} to 22·4{\%} (n=6 per group, p",
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AU - Hua, Zichun

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AU - Alkayed, Nabil

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