Jian Cheng[a], Patricia D. Hurn[a,b,c,1]

[a] Department ofAnesthesiology and Perioperative Medicine, UHS-2 3181 SW Sam Jackson Park Rd., Portland, OR 97239-3098, United States [b] Department of Physiology and Pharmacology, Portland, OR 97239, United States [c] Department ofNeurology Oregon Health and Science University, Portland, OR 97239, United States

[1]OHSU Research Center for Gender Based Medicine School ofMedicine Oregon Health; Science University 3181 SW Samjackson Pk Rd UHN-2 Portland OR 97239-3098, USA; E-mail: [email protected].

Abstract

Biologic sex and sex steroids are important factors in clinical and experimental stroke. This review evaluates key evidence that biological sex strongly alters mechanisms and outcomes from cerebral ischemia. The role of androgens in male stroke is understudied and important to pursue given that male sex is a well known risk factor for human stroke. To date, male sex steroids remain largely evaluated at the bench rather than the bedside. We review recent advances in our understanding of androgens in the context of ischemic cell death and neuroprotection. We also highlight some possible molecular mechanisms by which androgens impact ischemic outcomes.

Keywords: Sex; Sexual dimorphism Cerebral ischemia Androgens Stroke;

1.Stroke: a sexually dimorphic disease

We have only recently come to understand that stroke or “Brain attack” is a sexually dimorphic disease, i.e. the outcomes and mech-anisms of this form ofbrain injury are strongly linked to biological sex. Male sex is a well-acknowledged risk factor for human stroke, although little is known about the mechanism( s) behind this obser-vation. The overall incidence of stroke is higher in men vs. women in most nations, a trend that cuts across ethnic background [1]. This sexually dimorphic epidemiology is present in children [2] and persists until ages well beyond the menopausal years [3,4], suggesting that sex steroids are not the controllers of male vs. female risk for disease. For example, stroke rates in women do not equalize to those of men until beyond 75 years of age [4]. However, stroke risk does increase with age in both sexes, and there is some evidence that outcome from an ischemic event is worse in aged women than in their male counterparts (for reviews [5,6]). Therefore, knowledge of mechanisms of ischemic cell death and neuroprotection is important for both sexes. As discussed below, recent data suggest that ischemic pathobiology is strongly influenced by sex in experimental animal models, and cellular and molecular death mechanisms may not be identical in the male vs. female (for additional recent reviews [7-9]).

2.Sex and animal stroke models

Animal models have been used to evaluate side-by-side effects of cerebral ischemia, and in most reports, there are clear sex differences in outcomes [10-14]. Early evidence in female vs. male spontaneously hypertensive, genetically stroke prone rats uncovered the male phenotype of “ischemia-sensitivity” [15]. This landmark study of 2000 animals showed that life expectancy is longer in the female rat, and the development of spontaneous stroke lesions is delayed until an advanced age [15]. Female rats and mice of various inbred and outbred strains experience smaller tissue damage for an equivalent insult from focal or global cerebral ischemia and improved functional outcomes [10,11,14,16]. Similarly, male animals sustain greater injury than age-matched females after traumatic brain injury [17]. Even in rodent models carrying genetic risk factors associated with human stroke, e.g. insulin-dependent genetic diabetes [18], non-insulin dependent diabetes [19] and hypertension [10], females are less sensitive to cerebral ischemia than males. However, it should be noted that the majority of these studies have been carried out in rodents that are young (3-4 months) or middle aged (12-14 months). Whether sex differences reverse with age is not clear, although one recent study suggests that ischemic damage increases with age in female, but not male, mice [20]. Nevertheless, the data in aggregate emphasize that sex shapes outcome in experimental brain injury models, mimicking the epidemiology of human stroke.

3.XX vs. XY cells respond differently to injury

In vitro data directly support the related concepts that cell death after injury is sexually dimorphic and that some molecular injury and survival mechanisms are sex-specific. The latter concept has been formulated by studies of male vs. female cell cultures grown without background steroids. In early observations, female dopaminergic neurons were shown to tolerate exposure to toxic dopamine concentrations and survive twofold relative to male cells [21]. Similarly, female neurons from the cortical plate orventricular zone have greater longevity in culture than male cells, and differentially express higher levels of phosphorylated kinases such as Akt [22]. Sensitivity to glutamate, peroxynitrate (ONOO) and stau- rosporine in neuronal culture is sex-specific, with male neurons being more susceptible to glutamate and ONOO than females. In contrast, response to oxidants such as hydrogen peroxide (H2O2) is independent of cell sex [23].

These observations are not limited to neurons. Similar sex speci-ficity is present in the astrocytic response to oxygen and glucose deprivation (OGD), an in vitro model of “ischemia”, and to toxins that stimulate cell death pathways. Astrocytes are important supportive cells for neurons and brain vascular components in normal brain functioning. The inflammatory response of astrocytes and other glial cells, known as reactive gliosis, is a key component of the brain’s response to injury and neurodegenerative conditions. In cortical astrocytes, cultured from rat pups and segregated by sex, we have observed that female astrocytes are more resistant to OGD as compared to male cells, but sustain greater cell death when inflammatory mediators are combined with OGD compared to OGD alone [24]. Female astrocyte resistance to OGD is, in part, mediated by their ability to engage the endogenous enzyme P450 aromatase, metabolize 17β estradiol from androgen precursors, and capitalize on estradiol’s cytoprotective properties [25]. To verify the importance of sex differences in P450 aromatase function after OGD, we developed a novel method to establish sex-specific and genotype- specific single pup primary astrocyte cultures from wild-type and aromatase knockout (ArKO) mice [25]. Using this technique, we showed that female astrocytes lacking the P450 aromatase gene were highly sensitive to OGD, unlike their wild-type counterparts.

Furthermore, sex differences in astrocytic sensitivity to OGD were largely absent in ArKO cells, confirming that aromatase and sex- specific hormone production is an important basis for astrocytic sexual dimorphism under injury conditions.

4.Sex-specific ischemic cell death mechanisms

Emerging evidence suggests that the molecular signaling pathways engaged by cerebral ischemia in vivo or by cytotoxin administration to cultured cells are not identical in male vs. female brain. Data from genetically engineered mouse strains established the current working hypothesis that sexual dimorphism in ischemia is partly due to the genetic complement of cells, rather than solely to hormonal environment. When both sexes of genetic knockout mice are studied, one can readily observe if the gene of interest acts in an overtly sex-dependent way. Several genetic mechanisms not linked to sexual development or function have been shown to act dimorphically under injury conditions (Table 1). For example, neuronal nitric oxide synthase (nNOS) is well known to play an important role in neuronal death by fueling nitric oxide toxicity, peroxynitrite formation and protein nitration. Genetic deletion or pharmacological inhibition of nNOS is neuroprotective in the male, but not female, brain [14] or in hippocampal slice preparation [26].

Table 1. Sex-specific cell death mechanisms in ischemic brain injury.

To date, one of the best studied sex-specific mechanisms involves a set of molecules that lead to neuronal apoptosis after ischemic injury. Apoptosis, originally referred to as programmed cell death, is a slower form of post-ischemic pathology than frank tissue necrosis and so has received considerable interest as part of the search for human neuroprotective therapies. Apoptotic pathology involves at least two signaling cascades, one initiated through intrinsic, mitochondria-mediated mechanisms involving cytochrome C release, apoptosome assembly and caspase cleavage, and hence is known as a “caspase-dependent” pathway. An alternative caspase-independent pathway is triggered by post-ischemic DNA damage and has been recently named “parthanatos” (for recent review [27]). This caspase-independent pathway involves the activation of poly-ADP ribose polymerase (PARP), release of apoptosis-inducing factor (AIF) from the mitochondria and translocation of AIF to the nucleus to induce chromatin condensation, and large scale DNA fragmentation. PARP is a DNA repair enzyme that is critical to the caspase-independent death signaling that occurs after excitotoxic or ischemic insults in male mice or mixed sex cell culture [28]. Genetic deletion or pharmacological inhibition of PARP improves brain outcomes from cerebral ischemia in males [28], but not in females regardless of ovarian hormone status [14]. Similar sex-specificity has been reported in the developing brain treated with hypoxia-ischemia [29,30]. These early observations provided initial clues that the PARP-AIF death pathway may be highly engaged in male ischemic brain, less so in the female. Subsequent work has confirmed and expanded our understanding that the nNOS-PARP-AIF pathway is an important target in male cerebral ischemic pathology, while the intrinsic, caspase-dependent pathway is vital to female neuronal death. The therapeutic implication is that therapy designed to block nNOS/PARP mechanisms are likely to benefit the male, while anti-caspase therapies will be most effective in the female. Recent evidence in animals is bearing out this hypothesis (Table 1 and Refs. [30-34]).

In summary, these findings suggestthatthe response to cerebral injury in vivo and in vitro is partially a function of the sex of the cell. Examples of molecules that have sexually dimorphic roles in cerebral ischemic cell death mechanisms include apoptosis-inducing factor [30], caspase 3 [31,32], PARP [14,26,29], NOS [14,35], glutathione [23], Akt [36], astrocytic aromatase [24,25], glial fibrillary acidic protein (GFAP) [37], angiotensin II type 2 receptor [38], and the soluble epoxide hydrolase (sEH) [39]. However, this in no way discounts the importance of gonadal steroids or brain-derived neurosteroids as modulators of oxidant, toxic and ischemic challenges to the brain. An important, but highly understudied, issue is how the male “ischemic-sensitive” phenotype is established and if androgens contribute to the sexual dimorphism of ischemic brain injury.

5.Androgens and cerebral ischemia

Consistent but sparse evidence suggests that male sex and androgens impact ischemic outcomes and mechanisms of brain damage [7,40]. It has been assumed that androgens are detrimental to ischemic pathobiology because (1) male sex is a known stroke risk factor and (2) male animals sustain greater histological damage after experimental stroke than females. However, low circulating testosterone levels have also been associated with higher stroke incidence and worse outcomes after stroke in men [41-44]. Importantly, androgen levels dramatically drop following both experimental and clinical stroke [44,45]. Thus, ischemia-induced androgen loss may be as important as the steady level of androgens prior to the ischemic insult. In bench studies that control androgen levels, the data are conflicting and indicate that androgens can protect or exacerbate ischemic damage. Moreover, our recent finding that androgens exhibit highly dose-dependent effects on ischemic outcomes in animals further complicates the issues that surround male sex steroids [45]. Table 2 summarizes available in vitro and in vivo data on androgen effects in various brain injury models.

Table 2. Androgen effects in brain injury models.

6.In vitro effects of androgens

The brain is a target tissue for androgens, and numerous in vitro studies have suggested that androgens have direct effects on the responses of neurons and glia to injury. These insults include oxidative stress, excitotoxicity, serum deprivation and amyloid β (αβ) exposure. In aggregate, androgens can exacerbate or protect against damage from these insults in vitro. For instance, testosterone increases glutamate neurotoxicity in HT22 neuronal cells at 10 μM [46] and amplifies AMPA/kainate receptor associated oligodendrocyte damage at 100nM [47]. Another recent study demonstrates that testosterone protects against or exacerbates damage from excitotoxicity in cultured primary neurons, depending on the steroid concentration. Testosterone at supra-physiological concentrations (10 μM) amplifies excitotoxic neuronal death induced by N-methyl-D-aspartic acid (NMDA), however it is protective at 10nM and has no effect at intermediate concentrations [48]. In this in vitro model, the neuroprotective testosterone acts indirectly, i.e. via aromatization into 17β estradiol. In the presence of aromatase inhibitors, testosterone shows toxicity. In other studies, testosterone directly protects cultured neurons, as does the potent non-aromatizable androgen receptor (AR) agonist, dihydrotestosterone (DHT). For example, 1 μM testosterone protects cultured cerebral granule cells from oxidative stress, presumably through AR signaling since protection is abolished by flutamide [49]. In cultured primary hippocampal neurons, both testosterone and DHT (1-100 nM) protect against α β-induced cell death, and protection is notattenuated byaromatase inhibition [50-52].These results are consistent with an earlier study showing that both testosterone and estradiol confer neuroprotection to human cortical neurons from toxic αβ peptide. Interestingly, the protection by androgens and estrogen is mediated byARand estrogen receptor, respectively [53]. Testosterone (4 nM) also protects human neurons from serum deprivation-induced apoptosis via androgenic signaling [54]. One interpretation of the apparent paradox of beneficial vs. detrimental effects of androgens is that two potentially competing signaling mechanisms are engaged in vitro. For instance, in cultured astrocytes, activation of a putative membrane-associated AR by DHT promotes cell death, while signaling via classic intracellular AR is associated with neuroprotection [55]. Clearly, further study is required to unravel the mechanisms underlying the shift between androgen protective and damaging effects. Nevertheless, currently available in vitro studies have suggested that androgens can exhibit neuroprotective effects mechanistically independent from those of estrogen signaling.

7.Androgen actions in vivo

Consistent with the field’s initial assumption that androgens play a deleterious role in cerebral ischemia, some studies show that androgen treatment in castrated male rodents increases histological damage [46,56]. Stressors that reduce testosterone levels, e.g. anesthesia administered before the onset of cerebrovascular occlusion, improve ischemic outcomes in the male [57]. Yet, androgens administered after experimental stroke accelerate functional recovery after stroke [58]. This latter finding is consistent with clinical data showing that low plasma testosterone in men is inversely associated with stroke severity, infarct size and functional recovery [43]. Similarly, lower testosterone is associated with higher incidence of stroke in men [41]. In contrast to the assumed deleterious role of androgen, these studies support the hypothesis that androgens can be neuroprotective in cerebral ischemia. We have recently shown that maintaining testosterone or DHT plasma levels within the low physiological range throughout an episode of focal cerebral ischemia confers protection to both adult castrates (Fig. 1) and gonadally intact aged animals with naturally declining andro 59]. The benefit of controlled androgen availability has now been demonstrated in both rats and mice, and is mediated via direct AR signaling since neuroprotection is abrogated by flutamide [45]. Using the same hormone implantation technique, we also observed that high but physiologically relevant androgen levels exacerbate ischemic damage in castrated males [45,60], suggesting that androgens and androgen availability also have complex dose- dependent effects in vivo. To evaluate potential mechanisms behind androgens’ ability to enhance ischemic damage, we used microarray and real time PCR to identify gene candidates induced by DHT [60]. At high but physiologically relevant doses, DHT enhanced pro-inflammatory gene expression after middle cerebral artery occlusion, suggesting that enhanced inflammation may contribute to the deleterious effects of DHT. (For full list of genes identified by Affimetrix microarray in DHT treated castrated rats, see Ref. [60].)

Figure 1. Dose-dependent effects of DHT in experimental stroke. Low physiological doses of DHT decrease, while high but physiologically relevant doses of DHT increase, infarct volumes in young adult castrated male mice following middle cerebral artery occlusion. Infarct volumes are expressed as percentages of contralateral structures in castrated mice (CAST, n = 20) and in castrates implanted with 0.5 mg DHT [DHT(0.5), n =11] or 1.5mg DHT [DHT(1.5), n = 12]. Values are mean±SEM. *P < 0.05. Adapted from data as published in Ref. [45].

8.Neuroprotective mechanisms of androgens

Despite the complexities of androgenic dose-response relationships in ischemia, their neuroprotective properties remain of interest. AR expression has been confirmed in neurons throughout the brain, including cortical and striatal regions impacted by our focal ischemia models [61,62], therefore AR-regulated transcription is a potential mechanism underlying androgen neuroprotection. Although definitive characterization of genes that participate in androgen’s neuroprotection has not yet been accomplished, antioxidant proteins and stress-induced heat shock protein HSP70 have been implicated [49,53,63-65]. For example, testosterone protects cerebral granule cells against oxidative stress in an AR-dependent manner associated with a twofold increase in catalase activity [49]. Androgens also increase HSP70 expression after α β induced neurotoxicity [53]. Androgen non-genomic, rapid signaling pathways also have been implicated in androgen neuroprotection in cerebral ischemia. For example, androgen-induced activationofAkt protects cortical astrocytes against oxidative stress [55] and activation of mitogen-activated protein kinases (MAPK) protects hippocampal neurons against cell death induced by α β neurotoxicity [51]. In astrocytes and glial cell lines, DHT protection is associated with the activation of phosphoinositide-3 kinase (PI3K)/Akt as well as MAPK, while cell membrane impermeable DHT-BSA conjugates suppress MAPK and Akt activation and increase cell death [55,66]. These data suggest that intracellular AR- dependent activation of protective kinase signaling is important to DHT’s actions. Lastly, recent studies emphasize that androgens can rapidly but sustainably activate cAMP response element bind tissues [67-70]. Collectively, these results implicate a possible role of androgen activated non-genomic pathways in androgen neuroprotection following cerebral ischemia. Continued investigation of non-genomic signaling may provide important insights into the mechanisms underlying androgen neuroprotection.

In summary, androgens are clearly important but currently understudied steroid actors that are relevant to stroke and may advance our understanding of male vulnerability to cerebrovascular insults. Despite the complexity of androgen actions in vitro and in vivo, current clinical observations and limited animal data suggest that physiological levels of androgens exert neuroprotection in the male brain. The recently identified dose-dependent effects of androgens and the potential importance ofischemic stress-induced androgen loss may account for some controversial results in experimental stroke. Whether deleterious or beneficial, it is already clear that pleiotrophic androgens will impact multiple cell types of the injured brain and act through multiple molecular pathways in cerebral ischemia.

9.Sex on the brain in stroke: an opportunity

In conclusion, accumulatingevidence strongly suggests that bio-logical sex and sex steroids shape both the outcomes of cerebral ischemia and attendant cell death mechanisms. Understanding the differences between male and female cell responses to injury and repair offers an enormous opportunity to design and refine our therapeutic targets in a manner beneficial to both sexes. In addition, the use of animal and cell models that allow determination of sex differences, as well as the sex-specific scaffolding on which steroids operate, will allow us to understand experimental data in novel ways. Instead of minimizingorexcluding sex and sex steroids, as is frequently donein translational disease research, there is much to be gained by elucidating such fundamental mechanisms.

Acknowledgements

Support forthis research was provided by NIH grants NS049210, NR003521, the Bugher Foundation with the American Heart Asso-ciation and American Heart Association Grant 0825526G.

References

Sudlow CL, Warlow CP. Comparable studies of the incidence of stroke and its pathological types: results from an international collaboration. International Stroke Incidence Collaboration. Stroke 1997;28:491-9. 

Golomb MR, Fullerton HJ, Nowak-Gottl U, Deveber G, International Pediatric Stroke Study Group. Male predominance in childhood ischemic stroke: findings from the international pediatric stroke study. Stroke 2009;40:52-7. 

Giroud M, Milan C, Beuriat P, Gras P, Essayagh E, Arveux P, et al. Incidence and survival rates during a two-year period of intracerebral and subarachnoid haemorrhages, cortical infarcts, lacunes and transient ischaemic attacks. The Stroke Registry of Dijon: 1985-1989.Int J Epidemiol 1991;20:892-9. 

Sacco RL, Boden-Albala B, Gan R, Chen X, Kargman DE, Shea S, et al. Stroke incidence among white, black, and Hispanic residents of an urban community: the Northern Manhattan Stroke Study. AmJ Epidemiol 1998;147:259-68. 

Reeves MJ, Bushnell CD, Howard G, Gargano JW, Duncan PW, Lynch G, et al. Sex differences in stroke: epidemiology, clinical presentation, medical care, and outcomes. Lancet Neurol 2008;7:915-26. 

Appelros P, Stegmayr B, Terent A. Sex differences in stroke epidemiology: a systematic review. Stroke 2009;40:1082-90. 

LiuM, Dziennis S,Hum PD, Alkayed NJ. Mechanisms of gender-linked ischemic brain injury. Restor Neurol Neurosci 2009;27:163-79. 

Vagnerova K, Koerner IP, Hurn PD. Gender and the injured brain. Anesth Analg 2008;107:201-14. 

LangJT, McCullough LD. Pathways to ischemic neuronal cell death: are sex differences relevant? J Transl Med 2008;6:33. 

Alkayed NJ, Harukuni I, Kimes AS, London ED, Traystman RJ, Hurn PD. Gender- linked brain injury in experimental stroke. Stroke 1998;29:159-65 [discussion 166].  

I. Cheng, P.D. Hurn/Steroids xxx (2009) xxx-xxx  

Carswell HV, Anderson NH, Clark JS, Graham D, Jeffs B, Dominiczak AF, et al. Genetic and gender influences on sensitivity to focal cerebral ischemia in the stroke-prone spontaneously hypertensive rat. Hypertension 1999;33:681-5. 

Cai H, Yao H, Ibayashi S, Uchimura H, Fujishima M. Photothrombotic middle cerebral artery occlusion in spontaneously hypertensive rats: influence of sub-strain, gender, and distal middle cerebral artery patterns on infarct size. Stroke 1998;29:1982-6 [discussion 1986-7]. 

LiX, Blizzard KK, Zeng Z, DeVries AC, Hum PD, McCullough LD. Chronic behavioral testing after focal ischemia in the mouse: functional recovery and the effects of gender. Exp Neurol 2004;187:94-104. 

McCullough LD, Zeng Z, Blizzard KK, Debchoudhury I, Hurn PD. Ischemic nitric oxide and poly (ADP-ribose) polymerase-1 in cerebral ischemia: male toxicity, female protection.] Cereb Blood Flow Metab 2005;25:502-12. 

Yamori Y, Horie R, Handa H, Sato M, Fukase M. Pathogenetic similarity of strokes in stroke-prone spontaneously hypertensive rats and humans. Stroke 1976;7:46-53. 

Alkayed NJ, Murphy SJ, Traystman RJ, Hurn PD, Miller VM. Neuroprotective effects of female gonadal steroids in reproductively senescent female rats. Stroke 2000;31:161-8. 

Bramlett HM, Dietrich WD. Neuropathological protection after traumatic brain injury in intact female rats versus males or ovariectomized females. J Neuro-trauma 2001;18:891-900. 

Toung TK, Hurn PD, Traystman RJ, Sieber FE. Estrogen decreases infarct size after temporary focal ischemia in a genetic model of type 1 diabetes mellitus. Stroke 2000;31:2701-6. 

Vannucci SJ, Willing LB, Goto S, Alkayed NJ, Brucklacher RM, Wood TL, et al. Experimental stroke in the female diabetic, db/db, mouse. J Cereb Blood Flow Metab 2001;21:52-60. 

Liu F, Yuan R, Benashski SE, McCullough LD. Changes in experimental stroke outcome across the life span.J Cereb Blood Flow Metab 2009;29:792-802. 

Lieb K, AndraeJ, Reisert I, Pilgrim C. Neurotoxicity of dopamine and protective effects of the NMDA receptor antagonist AP-5 differ between male and female dopaminergic neurons. Exp Neurol 1995;134:222-9. 

Zhang L, Li PP, Feng X, BarkerJL, Smith SV, Rubinow DR. Sex-related differences in neuronal cell survival and signaling in rats. Neurosci Lett 2003;337:65-8. 

Du L, Bayir H, Lai Y, Zhang X, Kochanek PM, Watkins SC, et al. Innate gender-based proclivity in response to cytotoxicity and programmed cell death pathway.J Biol Chem 2004;279:38563-70. 

Liu M, Hurn PD, Roselli CE, Alkayed NJ. Role of P450 aromatase in sex-specific astrocytic cell death.J Cereb Blood Flow Metab 2007;27:135-41. 

Liu M, Oyarzabal EA, Yang R, Murphy SJ, Hurn PD. A novel method for assessing sex-specific and genotype-specific response to injury in astrocyte culture. J Neurosci Methods 2008;171:214-7. 

LiH, Pin S, Zeng Z, Wang MM, Andreasson KA, McCullough LD. Sex differences in cell death. Ann Neurol 2005;58:317-21. 

Wang Y, Dawson VL, Dawson TM. Poly(ADP-ribose) signals to mitochondrial AIF: a key event in parthanatos. Exp Neurol 2009;218:193-202. 

Eliasson MJ,Sampei K, MandirAS, Hurn PD,Traystman RJ, BaoJ, et al. Poly(ADP- ribose) polymerase gene disruption renders mice resistant to cerebral ischemia. NatMed 1997;3:1089-95. 

Hagberg H, Wilson MA, Matsushita H, Zhu C, Lange M, Gustavsson M, et al. PARP-1 gene disruption in mice preferentially protects males from perinatal brain injury.J Neurochem 2004;90:1068-75. 

Zhu C, Xu F, Wang X, Shibata M, Uchiyama Y, Blomgren K, et al. Different apoptotic mechanisms are activated in male and female brains after neonatal hypoxia-ischaemia.J Neurochem 2006;96:1016-27. 

Renolleau S, Fau S, Goyenvalle C, Joly LM, Chauvier D, Jacotot E, et al. Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7rat:a role for gender.J Neurochem 2007;100:1062-71. 

Liu F, Li Z, Li J, Siegel C, Yuan R, McCullough LD. Sex differences in caspase activation after stroke. Stroke 2009;40:1842-8. 

Yuan M, Siegel C, Zeng Z, Li J, Liu F, McCullough LD. Sex differences in the response to activation of the poly (ADP-ribose) polymerase pathway after experimental stroke. Exp Neurol 2009;217:210-8. 

Li J, McCullough LD. Sex differences in minocycline-induced neuroprotection after experimental stroke.J Cereb Blood Flow Metab 2009;29:670-4. 

Park EM, Cho S, Frys KA, Glickstein SB, Zhou P, AnratherJ, et al. Inducible nitric oxide synthase contributes to gender differences in ischemic brain injury. J Cereb Blood Flow Metab 2006;26:392-401. 

Kitano H, Young JM, Cheng J, Wang L, Hurn PD, Murphy SJ. Gender-specific response to isoflurane preconditioning in focal cerebral ischemia.J Cereb Blood Flow Metab 2007;27:1377-86. 

Cordeau Jr P, Lalancette-Hebert M, Weng YC, Kriz J. Live imaging of neuroin-flammation reveals sex and estrogen effects on astrocyte response to ischemic injury. Stroke 2008;39:935-42. 

Sakata A, Mogi M, Iwanami J, Tsukuda K, Min LJ, Fujita T, et al. Sex-different effect of angiotensin II type 2 receptor on ischemic brain injury and cognitive function. Brain Res 2009. Epub Aug 31 ; doi:10.1016. 

Zhang W, Iliff JJ, Campbell CJ, Wang RK, Hurn PD, Alkayed NJ. Role of soluble epoxide hydrolase in the sex-specific vascular response to cerebral ischemia. J Cereb Blood Flow Metab 2009;29:1475-81. 

Herson PS, Koerner IP, Hurn PD. Sex, sex steroids, and brain injury. Semin Reprod Med 2009;27:229-39. 

Yeap BB, Hyde Z, Almeida OP, Norman PE, Chubb SA, Jamrozik K, et al. Lower testosterone levels predict incident stroke and transient ischemic attack in older men.J Clin Endocrinol Metab 2009;94:2353-9. 

Hollander M, Koudstaal PJ, Bots ML, Grobbee DE, Hofman A, Breteler MM. Inci-dence, risk, and case fatality of first ever stroke in the elderly population. The Rotterdam Study.J Neurol Neurosurg Psychiatry 2003;74(3):317-21. 

Jeppesen LL, Jorgensen HS, Nakayama H, Raaschou HO, Olsen TS, Winther K. Decreased serum testosterone in men with acute ischemic stroke. Arterioscler ThrombVasc Biol 1996;16:749-54. 

Dash RJ, Sethi BK, Nalini K, Singh S. Circulating testosterone in pure motor stroke. Funct Neurol 1991 ;6:29-34. 

Uchida M, Palmateer JM, Herson PS, Devries AC, Cheng J, Hurn PD. Dose- dependent effects of androgens on outcome after focal cerebral ischemia in adult male mice.J Cereb Blood Flow Metab 2009;29:1454-62. 

Yang SH, Perez E, CutrightJ, Liu R,HeZ, DayAL, et al.Testosterone increases neu-rotoxicity of glutamate in vitro and ischemia-reperfusion injury in an animal model.J Appl Physiol 2002;92:195-201. 

CarusoA, Di Giorgi GereviniV, Castiglione M, Marinelli F,TomassiniV, Pozzilli C, et al. Testosterone amplifies excitotoxic damage of cultured oligodendrocytes. J Neurochem 2004;88:1179-85. 

Orlando R, Caruso A, Molinaro G, Motolese M, Matrisciano F, Togna G, et al. Nanomolarconcentrations of anabolic-androgenic steroids amplify excitotoxic neuronal death in mixed mouse cortical cultures. Brain Res 2007;1165:21-9. 

Ahlbom E, Prins GS, Ceccatelli S. Testosterone protects cerebellar granule cells from oxidative stress-induced cell death through a receptor mediated mecha-nism. Brain Res 2001 ;892:255-62. 

Pike CJ. Testosterone attenuates beta-amyloid toxicity in cultured hippocampal neurons. Brain Res 2001 ;919:160-5. 

Nguyen TV, Yao M, Pike CJ. Androgens activate mitogen-activated protein kinase signaling: role in neuroprotection. J Neurochem 2005;94: 1639-51. 

Nguyen TV, Yao M, Pike CJ. Flutamide and cyproterone acetate exert agonist effects: induction of androgen receptor-dependent neuroprotection. Endocrinology 2007;148:2 936-43. 

Zhang Y, Champagne N, Beitel LK, GoodyerCG, Trifiro M, LeBlancA. Estrogen and androgen protection ofhuman neurons against intracellular amyloid beta1-42 toxicity through heat shock protein 70. J Neurosci 2004;24:5315-21. 

Hammond J, Le Q, Goodyer C, Gelfand M, Trifiro M, LeBlanc A. Testosterone-mediated neuroprotection through the androgen receptor in human primary neurons.J Neurochem 2001;77:1319-26. 

Gatson JW, Singh M. Activation of a membrane-associated androgen receptor promotes cell death in primary cortical astrocytes. Endocrinology 2007;148:2458-64. 

Hawk T, Zhang YQ, Rajakumar G, Day AL, Simpkins JW. Testosterone increases and estradiol decreases middle cerebral artery occlusion lesion size in male rats. Brain Res 1998;796:296-8. 

Yang SH, Liu R, Wen Y, Perez E, CutrightJ, Brun-Zinkernagel AM, et al. Neu-roendocrine mechanism for tolerance to cerebral ischemia-reperfusion injury in male rats.J Neurobiol 2005;62:341-51. 

Pan Y, Zhang H, Acharya AB, Patrick PH, Oliver D, Morley JE. Effect of testos-terone on functional recovery in a castrate male rat stroke model. Brain Res 2005;1043:195-204. 

ChengJ, Hu W, Toung TJ, Zhang Z, Parker SM, Roselli CE, et al. Age-dependent effects of testosterone in experimental stroke. J Cereb Blood Flow Metab 2009;29:486-94. 

ChengJ, Alkayed NJ, Hurn PD. Deleterious effects of dihydrotestosterone on cerebral ischemic injury.J Cereb Blood Flow Metab2007;27:1553-62. 

Patchev VK, Schroeder J, Goetz F, Rohde W, Patchev AV. Neurotropic action of androgens: principles, mechanisms and novel targets. Exp Gerontol 2004;39:1651-60. 

DonCarlos LL, Sarkey S, Lorenz B, Azcoitia I, Garcia-Ovejero D, Huppenbauer C, et al. Novel cellular phenotypes and subcellular sites for androgen action in the forebrain. Neuroscience 2006;138:801-7. 

Lee SH, Kim M, Yoon BW, Kim YJ, Ma SJ, RohJK, et al. Targeted hsp70.1 disrup-tion increases infarction volume after focal cerebral ischemia in mice. Stroke 2001;32:2905-12. 

Rajdev S, Hara K, Kokubo Y, Mestril R, Dillmann W, Weinstein PR, et al. Mice overexpressing rat heat shock protein 70 are protected against cerebral infarc-tion. Ann Neurol 2000;47:782-91. 

Chisu V, Manca P, Zedda M, Lepore G, Gadau S, Farina V. Effects of testosterone on differentiation and oxidative stress resistance in C1300 neuroblastoma cells. Neuro Endocrinol Lett 2006;27:807-12. 

Gatson JW, Kaur P, Singh M. Dihydrotestosterone differentially modulates the mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt path-ways through the nuclear and novel membrane androgen receptor in C6 cells. Endocrinology2006;147:2028-34. 

Kousteni S, Han L, Chen JR, Almeida M, Plotkin LI, Bellido T, et al. Kinase-mediated regulation of common transcription factors accounts for the bone-protective effects of sex steroids. J Clin Invest 2003 ;111: 1651-64. 

Unni E, Sun S, Nan B, McPhaul MJ, Cheskis B, Mancini MA, et al. Changes in androgen receptor nongenotropic signaling correlate with transition of LNCaP cells to androgen independence. Cancer Res 2004;64:7156-68. 

Fix C, Jordan C, Cano P, Walker WH. Testosterone activates mitogen-activated protein kinase and the cAMP response element binding protein transcription factor in Sertoli cells. Proc Natl Acad Sci USA 2004;101:10919-24. 

Pike CJ, Nguyen TV, Ramsden M, Yao M, Murphy MP, Rosario ER. Androgen cell signaling pathways involved in neuroprotective actions. Horm Behav 2008;53:693-705.