Estradiol is protective against ischemic brain damage through signal trasducer and activator of transcription 3 (STAT3)

Suzan Dziennis, Taiping Jia, Oline Ronnekleiv, Patricia D. Hurn, Nabil Alkayed

Research output: Contribution to journalArticle

Abstract

Background and Aims: Estradiol is neuroprotective in animal models of cerebral ischemia (1). Although the precise mechanisms are unknown, estrogen's beneficial properties are in part linked to rapid activation of signal transduction pathways that lead to the transcription of neuroprotective genes (2). Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated in response to both estrogen (3) and ischemia (4). Upon activation, STAT3 translocates to the nucleus where it upregulates neuroprotective genes, such as bcl-2 (5). We wished to determine whether estradiol replacement enhances STAT3 activation in female rat brain after focal cerebral ischemia and if STAT3 activation plays a protective role against ischemic brain injury. Methods: Adult female rats were ovariectomized (OVX) at 10-12 weeks and implanted with 25 μg 17s-estradiol (EST) 7 days prior to middle cerebral artery occlusion (MCAO). A 2-hour MCAO was induced by the intraluminal filament technique. Laser-Doppler cerebrocortical perfusion, mean arterial pressure, PO2, PCO2, pH, glucose and terminal plasma estrogen were measured. A single intraperintoneal injection of the STAT3 inhibitor JSI-124 (1mg/kg, was given 15 minutes after MCAO. Brains from separate groups were removed and processed for one of the following analyses: 1) brains were subdissected and rapidly frozen to prepare cortical nuclear and cytosolic extracts for phospho-STAT3 (P-STAT3) and total-STAT3 (T-STAT3) immunoblot analysis, 2) brains were sectioned and stained with 2,3,5-triphenyltetrazolium chloride (TTC) for infarct size measurement, or 3) brains were fixed in 4% paraformaldehyde for immunohistochemistry (IHC) with P-STAT3, Bcl-2 and MAP2 antibodies. Phospho-STAT3 was measured at 3 and 22 hours post reperfusion. IHC and infarct volume were measured at 22 hours post reperfusion. Results: Physiological variables were not different among groups. Estradiol increased P-STAT3 in the cytosolic and nuclear fractions at 3 hours following MCAO. In estradiol replaced animals, P-STAT3 remained elevated in nuclear fractions at 22 hours in ischemic cortex. Furthermore, the nuclear P-STAT3 co-localized with cells expressing neuronal and survival markers MAP-2 and Bcl-2. The P-STAT3 inhibitor JSI-124 abolished the effect of estradiol to reduce infarct size after MCAO in ovariectomized rats. Conclusion: Estradiol enhances STAT3 phosphorylation in ischemic brain, and activated STAT3 contributes to the hormone's neuroprotective property.

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

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Estradiol
Middle Cerebral Artery Infarction
Brain
Estrogens
Brain Ischemia
Transcriptional Activation
Reperfusion
Immunohistochemistry
STAT3 Transcription Factor
Brain Injuries
Genes
Signal Transduction
Arterial Pressure
Lasers
Transcription Factors
Up-Regulation
Ischemia
Animal Models
Perfusion
Phosphorylation

ASJC Scopus subject areas

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

Cite this

Estradiol is protective against ischemic brain damage through signal trasducer and activator of transcription 3 (STAT3). / Dziennis, Suzan; Jia, Taiping; Ronnekleiv, Oline; Hurn, Patricia D.; Alkayed, Nabil.

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

Research output: Contribution to journalArticle

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title = "Estradiol is protective against ischemic brain damage through signal trasducer and activator of transcription 3 (STAT3)",
abstract = "Background and Aims: Estradiol is neuroprotective in animal models of cerebral ischemia (1). Although the precise mechanisms are unknown, estrogen's beneficial properties are in part linked to rapid activation of signal transduction pathways that lead to the transcription of neuroprotective genes (2). Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated in response to both estrogen (3) and ischemia (4). Upon activation, STAT3 translocates to the nucleus where it upregulates neuroprotective genes, such as bcl-2 (5). We wished to determine whether estradiol replacement enhances STAT3 activation in female rat brain after focal cerebral ischemia and if STAT3 activation plays a protective role against ischemic brain injury. Methods: Adult female rats were ovariectomized (OVX) at 10-12 weeks and implanted with 25 μg 17s-estradiol (EST) 7 days prior to middle cerebral artery occlusion (MCAO). A 2-hour MCAO was induced by the intraluminal filament technique. Laser-Doppler cerebrocortical perfusion, mean arterial pressure, PO2, PCO2, pH, glucose and terminal plasma estrogen were measured. A single intraperintoneal injection of the STAT3 inhibitor JSI-124 (1mg/kg, was given 15 minutes after MCAO. Brains from separate groups were removed and processed for one of the following analyses: 1) brains were subdissected and rapidly frozen to prepare cortical nuclear and cytosolic extracts for phospho-STAT3 (P-STAT3) and total-STAT3 (T-STAT3) immunoblot analysis, 2) brains were sectioned and stained with 2,3,5-triphenyltetrazolium chloride (TTC) for infarct size measurement, or 3) brains were fixed in 4{\%} paraformaldehyde for immunohistochemistry (IHC) with P-STAT3, Bcl-2 and MAP2 antibodies. Phospho-STAT3 was measured at 3 and 22 hours post reperfusion. IHC and infarct volume were measured at 22 hours post reperfusion. Results: Physiological variables were not different among groups. Estradiol increased P-STAT3 in the cytosolic and nuclear fractions at 3 hours following MCAO. In estradiol replaced animals, P-STAT3 remained elevated in nuclear fractions at 22 hours in ischemic cortex. Furthermore, the nuclear P-STAT3 co-localized with cells expressing neuronal and survival markers MAP-2 and Bcl-2. The P-STAT3 inhibitor JSI-124 abolished the effect of estradiol to reduce infarct size after MCAO in ovariectomized rats. Conclusion: Estradiol enhances STAT3 phosphorylation in ischemic brain, and activated STAT3 contributes to the hormone's neuroprotective property.",
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T1 - Estradiol is protective against ischemic brain damage through signal trasducer and activator of transcription 3 (STAT3)

AU - Dziennis, Suzan

AU - Jia, Taiping

AU - Ronnekleiv, Oline

AU - Hurn, Patricia D.

AU - Alkayed, Nabil

PY - 2007/11/13

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N2 - Background and Aims: Estradiol is neuroprotective in animal models of cerebral ischemia (1). Although the precise mechanisms are unknown, estrogen's beneficial properties are in part linked to rapid activation of signal transduction pathways that lead to the transcription of neuroprotective genes (2). Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated in response to both estrogen (3) and ischemia (4). Upon activation, STAT3 translocates to the nucleus where it upregulates neuroprotective genes, such as bcl-2 (5). We wished to determine whether estradiol replacement enhances STAT3 activation in female rat brain after focal cerebral ischemia and if STAT3 activation plays a protective role against ischemic brain injury. Methods: Adult female rats were ovariectomized (OVX) at 10-12 weeks and implanted with 25 μg 17s-estradiol (EST) 7 days prior to middle cerebral artery occlusion (MCAO). A 2-hour MCAO was induced by the intraluminal filament technique. Laser-Doppler cerebrocortical perfusion, mean arterial pressure, PO2, PCO2, pH, glucose and terminal plasma estrogen were measured. A single intraperintoneal injection of the STAT3 inhibitor JSI-124 (1mg/kg, was given 15 minutes after MCAO. Brains from separate groups were removed and processed for one of the following analyses: 1) brains were subdissected and rapidly frozen to prepare cortical nuclear and cytosolic extracts for phospho-STAT3 (P-STAT3) and total-STAT3 (T-STAT3) immunoblot analysis, 2) brains were sectioned and stained with 2,3,5-triphenyltetrazolium chloride (TTC) for infarct size measurement, or 3) brains were fixed in 4% paraformaldehyde for immunohistochemistry (IHC) with P-STAT3, Bcl-2 and MAP2 antibodies. Phospho-STAT3 was measured at 3 and 22 hours post reperfusion. IHC and infarct volume were measured at 22 hours post reperfusion. Results: Physiological variables were not different among groups. Estradiol increased P-STAT3 in the cytosolic and nuclear fractions at 3 hours following MCAO. In estradiol replaced animals, P-STAT3 remained elevated in nuclear fractions at 22 hours in ischemic cortex. Furthermore, the nuclear P-STAT3 co-localized with cells expressing neuronal and survival markers MAP-2 and Bcl-2. The P-STAT3 inhibitor JSI-124 abolished the effect of estradiol to reduce infarct size after MCAO in ovariectomized rats. Conclusion: Estradiol enhances STAT3 phosphorylation in ischemic brain, and activated STAT3 contributes to the hormone's neuroprotective property.

AB - Background and Aims: Estradiol is neuroprotective in animal models of cerebral ischemia (1). Although the precise mechanisms are unknown, estrogen's beneficial properties are in part linked to rapid activation of signal transduction pathways that lead to the transcription of neuroprotective genes (2). Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated in response to both estrogen (3) and ischemia (4). Upon activation, STAT3 translocates to the nucleus where it upregulates neuroprotective genes, such as bcl-2 (5). We wished to determine whether estradiol replacement enhances STAT3 activation in female rat brain after focal cerebral ischemia and if STAT3 activation plays a protective role against ischemic brain injury. Methods: Adult female rats were ovariectomized (OVX) at 10-12 weeks and implanted with 25 μg 17s-estradiol (EST) 7 days prior to middle cerebral artery occlusion (MCAO). A 2-hour MCAO was induced by the intraluminal filament technique. Laser-Doppler cerebrocortical perfusion, mean arterial pressure, PO2, PCO2, pH, glucose and terminal plasma estrogen were measured. A single intraperintoneal injection of the STAT3 inhibitor JSI-124 (1mg/kg, was given 15 minutes after MCAO. Brains from separate groups were removed and processed for one of the following analyses: 1) brains were subdissected and rapidly frozen to prepare cortical nuclear and cytosolic extracts for phospho-STAT3 (P-STAT3) and total-STAT3 (T-STAT3) immunoblot analysis, 2) brains were sectioned and stained with 2,3,5-triphenyltetrazolium chloride (TTC) for infarct size measurement, or 3) brains were fixed in 4% paraformaldehyde for immunohistochemistry (IHC) with P-STAT3, Bcl-2 and MAP2 antibodies. Phospho-STAT3 was measured at 3 and 22 hours post reperfusion. IHC and infarct volume were measured at 22 hours post reperfusion. Results: Physiological variables were not different among groups. Estradiol increased P-STAT3 in the cytosolic and nuclear fractions at 3 hours following MCAO. In estradiol replaced animals, P-STAT3 remained elevated in nuclear fractions at 22 hours in ischemic cortex. Furthermore, the nuclear P-STAT3 co-localized with cells expressing neuronal and survival markers MAP-2 and Bcl-2. The P-STAT3 inhibitor JSI-124 abolished the effect of estradiol to reduce infarct size after MCAO in ovariectomized rats. Conclusion: Estradiol enhances STAT3 phosphorylation in ischemic brain, and activated STAT3 contributes to the hormone's neuroprotective property.

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