Sex-Specific Role of Thioredoxin in Neuroprotection Against Iron-Induced Brain Injury Conferred by Estradiol
Background and Purpose— Accumulation of iron after intracerebral hemorrhage causes free radical formation and oxidative damage resulting in liquefaction. The aim of this study was the investigation of molecular mechanisms underlying estrogen-mediated neuroprotective effect against iron-induced brain injury in vivo.
Methods— Age-matched male and female Sprague-Dawley rats were stereotaxically infused with either ferrous citrate (FC) or saline (10 μL) into the right caudate nucleus. Beta-estradiol 3-benzoate (E2) capsule was implanted subcutaneously at 24 hours before infusion of FC. The severity of brain injury and neurological deficits were measured by histological quantification and forelimb asymmetry test, respectively. The role of thioredoxin (Trx) in E2-mediated neuroprotective effect was examined by intrastriatal administration of a Trx reductase inhibitor, 5,5-dithiobis-(2-nitrobenzoic acid), and small interfering RNA.
Results— FC induced greater brain injury in male rats than females. E2 treatment reduced FC-induced brain injury in both sexes. E2 significantly increased protein level and activity of Trx in the caudate nucleus of females but not males. Administration of female rats with 5,5-dithiobis-(2-nitrobenzoic acid) or Trx small interfering RNA to the caudate nucleus decreased the protective effect of E2 against FC-induced injury. The protein and mRNA levels of estrogen receptorα, but not estrogen receptorβ, were more abundant in the caudate nucleus of female rats.
Conclusions— Increase of brain Trx activity might play an important role in the E2-mediated neuroprotective effect against FC-induced brain injury in female rats. Understanding of the sex differences in the Trx-mediated neuroprotective effect by E2 might help in improving treatment of brain dysfunction after hemorrhagic stroke and/or head trauma.
Intracerebral hemorrhage (ICH) is caused by hypertension and related cerebral aneurysm and accounts for 10% to 30% of all stroke cases.1 ICH is associated with a high mortality rate, neurological deficits, and long-term disability. Reports indicate that females are less susceptible to stroke- and trauma-induced brain injury than age-matched males until reaching menopause.2,3 Animal studies of cerebral ischemia also indicate that female rats experience less neuronal damage than male rats,4 and estrogen treatment protects the brain from stroke in vivo.5,6 However, the underlying mechanisms remain to be elucidated. A recent in vitro study provided new evidence to support a possible role of thioredoxin (Trx) in mediating 17β-estradiol-mediated neuroprotection against brain injury.7
Although the underlying mechanism of ICH-induced brain injury is not fully elucidated,8 one theory holds that the extensive lysis of extravascular red blood cells in the brain after ICH lead to overproduction of ferrous iron, which undergoes a redox cycle, including Haber-Weiss reaction and/or a Fenton reaction in the presence of oxygen, citric acid, and isocitric acids.9 Redox cycling of iron complexes (ie, bidentate and tridentate ferrous citrate and hemoglobin) causes persistent conversion of oxygen into reactive oxygen species that lead to axonal dystrophy and cell death.10 Because deferoxamine, an iron chelator, reduces acute ICH-induced brain edema11 and improves functional outcomes after bleeding, a detrimental role of iron in ICH has been indicated.12 Accordingly, we established the present rat model to mimic the ICH-induced iron accumulation and oxidative stress by intrastriatal infusion of the small molecular weight iron complexes, ferrous citrate (FC).
Cellular antioxidative activities include induction of antiapoptotic or antioxidant molecules such as Trx13 and other survival proteins, which are crucial for reactive oxygen species scavenging and for preventing oxidative stress-induced apoptosis.14 Previous reports indicate that Trx induction mediates the preconditioning-induced neuroprotection15 and the β-estradiol 3-benzoate (E2)-mediated neuroprotection7 against serum deprivation-induced cell death in vitro. Accordingly, we hypothesize that Trx might mediate the neuroprotection conferred by E2 against iron-induced brain injury in vivo.
Materials and Methods
A total of 228 12-week-old male and female Sprague-Dawley rats (350 to 420 g; LASC; Charles River Technology, Taipei, Taiwan) were used. All procedures were approved by the Kaohsiung Medical University Committee for the Use of Experimental Animals.
Castration was performed under anesthesia with pentobarbital (35 mg/kg, intraperitoneally). A silastic tube (2-mm inner diameter, 30 mm in length; Shin-Etsu Polymer Co, Ltd) containing 1 mmol/L E2 (Sigma) was implanted subcutaneously at 24 hours before infusion of FC. A microinfusion pump (CMA Microdialysis) was used to infuse 10 μL FC (2.55 μmol/L; Sigma) into the right caudate nucleus (CN; coordinates: 0.2 mm anterior, 3.5 mm lateral, and 5.5 mm ventral to bregma) at a rate of 10 μL/min. Sham-operated and saline-injected rats were used as normal and vehicle control groups, respectively.
Forelimb Use Asymmetry Test
Individual rats were placed in a transparent cylinder (25 cm in diameter and 30 cm in height) in the dark, and the use of ipsilateral limbs (I), contralateral limbs (C), or simultaneous use of both forelimbs (B) was observed for a 5-minute period. The test was randomized, blind, and repeated twice in each rat. The forelimb use asymmetry ratio was calculated using the following equation: [I/(I+C+B)]−[C/(I+C+B)].16
The paraffin-embedded tissue was serially sectioned into 10 μm thickness. After hematoxylin and eosin staining, the severity of brain lesion in every fifth section of the CN was analyzed by Image-pro Plus software (Universal Imaging Corp) according to the staining intensity of the CN area. The total hemispheric volume was integrated from the hemispheric areas of 20 sections. The lesion ratio was estimated by dividing the hemispheric volume of the CN on the ipsilateral side by that on the contralateral side.
Western Blot Analysis
Equal amounts of protein from each sample were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membrane, incubated with antibody against Trx (1:200; Santa Cruz Laboratories), estrogen receptor (ER)α (1:1000; Upstate Biotechnology), or ERβ (1:500; Upstate Biotechnology) followed by goat anti-rabbit immunoglobulin G (1:5000, horseradish peroxidase conjugated; Santa Cruz Laboratories) and then visualized by enhanced chemiluminescence (Amersham).
The reductive activity of Trx was examined by measuring the number of sulfhydryl groups. The Ellman working reagent (Sigma) contains 50 μL of 2 mmol/L 5,5-dithiobis-(2-nitrobenzoic acid; DTNB) solution, 100 μL 1 mol/L Tris solution, and 840 μL distilled water. N-acetyl-l-cysteine was used to set a standard sulfhydryl calibration curve. Protein sample (100 μg/10 μL) was added to the 990 μL Ellman working reagent. Ten microliters of sample buffer was used as a control. The reaction mixture was then incubated for 5 minutes at 37°C, and optical absorbance at 412 nm was measured using an enzyme reader (BioTek Instrument) immediately after incubation.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA (5 μg) was transcribed to cDNA using random primers (Promega Corporation). The sequences of primers for ER-α, ER-β, and glyceraldehyde-3-phosphate dehydrogenase are: ERα: 5′-GCAGCGAGAAGGGAAACATC-3′ (forward) and 5′-GCATAGTCGTTACACACAGCACAG-3′ (reverse); ERβ: 5′-TTCTGGGCACCTGTCTCCTTTA-3′ (forward) and 5′-GGACTCTTTTGAGGTTCTGCATAGA-3′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase: 5′-CTCTACCCACGGCAAGTTCAA-3′ (forward) and 5′-GGAAGATGGTGATGGGTTTCC-3′ (reverse). The cDNA amplifications were carried out in a 25μL mixture containing 12.5 μL of 2× SYBR Green PCR Master Mix (Applied Biosystems), 200 nmol/L primers, and 20 ng cDNA. Thermal cycling was performed on the Applied Biosystems 7900HT real-time polymerase chain reaction system. Data were analyzed using SDS Version 2.1 software (Applied Biosystems).
Trx Small Interfering RNA
The Trx small interfering (si)RNA was synthesized by Ambion (Applied Biosystems Business). Two Trx siRNA sequences were used (Duplex 1 [5′→ 3′]: sense GCUCGAAGCCACUAUUACGtt, antisense CGUAAUAGUGGCUUCGAGCtt; Duplex 2: sense GGUUAAAACCUGU ACCUUUtt, antisense AAAGGUACAGGUUUUAACCtg). A 5μL (80 nmol/L) siRNA was mixed with 5 μL transfection reagent (Dharmacon), incubated for 20 minutes, and then infused into the right CN at 6 hours before infusion of FC. Dharmacon siCONTROL nontargeting siRNA (catalog number D-001206-13) was used as a negative control.
FC-induced injury was compared between brains from male and female specimens using 2-way analysis of variance followed by Scheffé post hoc test using SPSS software (Statistical Package for the Social Sciences). The data for the effects of E2, DTNB, or Trx siRNA on iron-induced brain injury and the Trx protein level and activity were analyzed by multiway analysis of variance using JMP Version 5.1.2 software (a business unit of SAS) to determine the effect of each factor and the interaction between 2 factors. Significance was accepted at P<0.05.
Protective Effect of E2 on FC-Induced Neurological Deficit and Brain Lesion
Neurological deficit was reflected by the higher forelimb use asymmetry ratio. Infusion of FC significantly increased the forelimb use asymmetry ratio in male and female rats (64%±11.47% and 34%±6.76%, respectively) and increased more in the ovariectomized rats as compared with intact females, but there was no significant difference between castrated and intact male rats. To confirm the success of ovariectomy or E2 implantation, the serum estradiol levels were measured by using an enzyme immunoassay kit. E2 implantation significantly reduced the forelimb use asymmetry ratio in male and female rats by 23%±11.7% and 26%±12.9%, respectively (Figure 1A). Comparisons of the CN injury showed that the ratio of lesions caused by infusion of FC in male rats (37.83%±9.55%) was significantly higher than that in female rats (11.54%±2.59%). Castration increased the FC-induced lesion ratio in female rats, but not males. The E2 implantation significantly decreased the ratio of FC-induced lesion in the CN of castrated male and female rats (13%±6.39% and 10%±5.01%, respectively; Figure 1B).
Sex Difference in Trx Protein Levels and Activities in the CN After Infusion of FC and E2 Implantation
The basal level of Trx protein in the CN was significantly higher in female rats than in males. Saline infusion did not change significantly the Trx protein level in the CN of either male or female rats, whereas infusion of FC significantly increased the protein level of Trx in male rats but not females. The Trx protein level in the CN did not differ between intact and castrated male rats but was significantly decreased in ovariectomized rats as compared with intact female rats. E2 implantation significantly increased the Trx level in FC-infused female but not male rats (Figure 2A). To evaluate the involvement of Trx in the neuroprotective effect of E2 on FC-induced brain injury, the reductive activity of Trx in the CN was examined. As shown in Figure 2B, infusion of FC significantly increased the reductive activity in the CN in male but not female rats. Castration did not change the reductive activity in the CN in male and female rats. Implantation of an E2 capsule significantly increased the reductive activity in the CN of FC-infused female rats but not males.
Involvement of Trx in the E2-Mediated Neuroprotection Against FC-Induced Brain Injury
Because E2-induced increase of Trx was observed only in FC-infused female rats, we further examined the role of Trx in E2-induced neuroprotection in female rats by intrastriatal administration of a Trx reductase inhibitor, DTNB. The DTNB per se did not significantly change the Trx protein level (data not shown). However, DTNB significantly decreased the effects of E2 on Trx activity (Figure 3A), forelimb use asymmetry ratio (Figure 3B), and lesion ratio (Figure 3C). Moreover, Trx siRNA decreased the Trx protein level in the CN (Figure 3D) and significantly decreased the protective effect of E2 on FC-induced behavioral deficit (Figure 3E) and brain lesion (Figure 3F).
Expression of ERα and ERβ in the CN of Female and Male Rats
To test whether ER is involved in the sex dimorphism of Trx induction caused by E2, we examined the levels of mRNA and protein of both ERα and ERβ in the CN. As shown in Figure 4, the expression levels of ERα mRNA (Figure 4A) and protein (Figure 4B) and the number of ERα-immunoreactive cells (Figure 4C) in the CN were higher in female than male brains. However, the levels of brain ERβ mRNA and protein in the CN of female rats were similar to those in male rats.
It has been suggested that Trx plays a critical role in E2-induced neuroprotection against oxidative neuronal injury in vitro.17 This in vivo study demonstrated that Trx induction may participate in E2-mediated neuroprotection against iron-induced brain injury in female, but not male, rats. The present study demonstrated that the E2-mediated protection against the FC-induced brain injury in female rats was reduced by intrastriatal pretreatment with siRNA againt Trx, which reduced the Trx protein levels (Figure 3D–F). Although the Trx reductive activity in the rats treated with siRNA Trx was not measured, the role of endogenous Trx in the brain was confirmed by using a Trx reductase inhibitor DTNB, which blocked the redox cycling of endogenous Trx and prevented the FC-induced brain injury in female rats (Figure 3A–C). These in vivo findings are consistent with the early in vitro findings using antisense oligonucleotide to decrease the Trx level and thus the Trx activity.17
Numerous clinical studies indicate that premenopausal women have lower risk in stroke than age-matched men, and estrogen improves stroke outcome after vascular occlusion in animal4 and human18 studies. However, the precise mechanisms underlying sex differences in functional outcomes after stroke are still unclear. A previous report showed that male rats with cortex lesions exhibited persistent water maze deficit throughout 10 days of test, whereas brain-damaged females did not,2 suggesting that females are less susceptible to injury after brain lesion than males. During the past decade, knowledge of the mechanisms underlying brain injury induced by ICH has rapidly accumulated. The coagulation cascade, inflammation, and breakdown of hemoglobin products (iron in particular)10 all contribute to ICH-induced injury.8 Because deferoxamine, an iron chelator, improved the functional outcome by reducing ICH-induced brain edema and atrophy, we applied the FC-infused rat model to examine the neuroprotective mechanism of E2. After the intrastriatal infusion of FC, both the forelimb use asymmetry ratio and lesion ratio showed significant differences between male and female rats (Figure 1). The FC-induced increases in forelimb use asymmetry ratio and lesion ratio in male rats (64.86% and 35.32%, respectively) were significantly higher than those observed in females (30.96% and 8.9%, respectively). Additionally, the ratios of forelimb use asymmetry ratio and lesion caused by intrastriatal infusion of FC were decreased in castrated male rats but were increased in ovariectomized rats. These results imply that the endogenous sex hormonal milieu might affect the severity of brain injury. The E2 implantation significantly protected the CN of both male and female rats against the FC-induced behavioral deficit and brain lesion. Accordingly, the rat model of brain injury induced by intrastriatal infusion of FC in this study provides a good model for studying sex-specific brain injury caused by iron overload and the protective mechanism conferred by E2.
Excessive ferrous iron enters a redox cycle and causes persistent reactive oxygen species production as well as oxidative stress, which induce survival proteins such as Trx to quench the stress. Many in vitro and in vivo models have confirmed the antioxidant effects of estrogen.5,19 Physiological concentrations of E2 (<10 nM) have been reported to upregulate Trx expression in vitro.7 This estrogen-mediated induction of Trx plays a pivotal role in the ER-mediated neuroprotection in human neuroblastoma cells.17 Estrogen can also provide neuroprotection through an ER-independent pathway.20,21 However, the exact neuroprotective mechanism of E2 under neuronal iron overload is still unclear. Surprisingly, the results from our present study showed that Trx induction by FC in the CN was significantly greater in male than female rats. A possible explanation is that a higher concentration of endogenous E2 in female rats can reduce the FC-induced increase of oxidant stress, which is a stimulant for Trx expression, through an ER-independent mechanism. Notably, the FC-induced increase of Trx was not significantly changed in E2-implanted castrated males but significantly increased in E2-implanted ovariectomized female rats. Because activation of ER can increase the Trx expression7 and more abundance of ERα was observed in the CN of female rats (Figure 4), a more prominent ER-dependent protective effect of estradiol may exist in female brains and sex differences in Trx induction in response to E2 implantation might be explained by the sex difference in the levels of ER in the CN. The involvement of ER isoforms in the estrogen-mediated Trx induction during basal physiological condition or under iron-induced stress has to be confirmed by further studies. Antagonist or siRNA of either ERα or ERβ may be applied to address this issue.
Stroke is recognized as a sexually dimorphic disease. Reports indicate that females have better free radical homeostasis and stronger defense capacity against oxidative brain damage as compared with males.22 Moreover, stress-induced HSP72 expression in the brain also exhibits sexual dimorphism.23 The present study revealed that the protein levels of Trx in the CN of female brains were greater than in males. Furthermore, Trx was found to be involved in the E2-mediated neuroprotection in a sex-dependent manner, which implies a sexual dimorphism in the molecular pathogenesis of iron-induced brain injury. The underlying mechanism of sex-specific neuroprotection requires further investigation to develop sex-specific therapeutic strategies for preventing brain dysfunction after hemorrhage.
E2 increased the Trx expression, which in turn protected the CN against FC-induced injury in female but not male rats. This evidence of sexual dimorphism in the Trx-mediated neuroprotective effect of E2 may be useful for developing sex-specific therapeutic strategies for preventing brain dysfunction caused by hemorrhage and/or by neurodegenerative disease associated with iron overload.
Sources of Funding
This work was supported by research grants from the National Science Council of Taiwan (NSC-95-2320-B-037-032-MY2 and NSC97-2314-B-037-039-MY2 to C.H.; NSC-95-2320-B-038-027-MY2 to C.C.C.).
- Received July 14, 2009.
- Revision received October 1, 2009.
- Accepted October 13, 2009.
Thrift AG, Donnan GA, McNeil JJ. Epidemiology of intracerebral hemorrhage. Epidemiol Rev. 1995; 17: 361–381.
Alkayed NJ, Harukuni I, Kimes AS, London ED, Traystman RJ, Hurn PD. Gender-linked brain injury in experimental stroke. Stroke. 1998; 29: 159–165;discussion 166.
Lee SY, Andoh T, Murphy DL, Chiueh CC. 17beta-estradiol activates ICI 182,780-sensitive estrogen receptors and cyclic GMP-dependent thioredoxin expression for neuroprotection. FASEB J. 2003; 17: 947–948.
Wu J, Hua Y, Keep RF, Nakamura T, Hoff JT, Xi G. Iron and iron-handling proteins in the brain after intracerebral hemorrhage. Stroke. 2003; 34: 2964–2969.
Hua Y, Schallert T, Keep RF, Wu J, Hoff JT, Xi G. Behavioral tests after intracerebral hemorrhage in the rat. Stroke. 2002; 33: 2478–2484.
Andoh T, Chock PB, Chiueh CC. The roles of thioredoxin in protection against oxidative stress-induced apoptosis in SH-SY5Y cells. J Biol Chem. 2002; 277: 9655–9660.
Rau SW, Dubal DB, Bottner M, Gerhold LM, Wise PM. Estradiol attenuates programmed cell death after stroke-like injury. J Neurosci. 2003; 23: 11420–11426.
Yi KD, Cai ZY, Covey DF, Simpkins JW. Estrogen receptor-independent neuroprotection via protein phosphatase preservation and attenuation of persistent extracellular signal-regulated kinase 1/2 activation. J Pharmacol Exp Ther. 2008; 324: 1188–1195.
Nilsen J, Diaz Brinton R. Mechanism of estrogen-mediated neuroprotection: regulation of mitochondrial calcium and BCL-2 expression. Proc Natl Acad Sci U S A. 2003; 100: 2842–2847.
Nickerson M, Kennedy SL, Johnson JD, Fleshner M. Sexual dimorphism of the intracellular heat shock protein 72 response. J Appl Physiol. 2006; 101: 566–575.