These experiments show several important new findings. First, E2 increases stroke-induced neurogenesis. This is not a sex-specific effect as both male and ovx female mice responded to E2. The proliferative effect of E2 was only unmasked under pathological conditions, in this case, stroke, as no increase was seen in E2-treated sham mice. Although E2 robustly increased ischemic neurogenesis, stroke itself remained a potent stimulus, as oil-treated mice of both sexes had significantly increased neurogenesis compared with sham animals. This may be secondary to increased BDNF levels after stroke. Second, BrdU+-labeled cells were present as long as 6 weeks after stroke and acquired a mature neuronal phenotype as indicated by NeuN colabeling. Third, E2-mediated enhancement of neurogenesis in the ischemic brain occurred in both neurogenic niches in the adult brain, the SVZ and DG. Stroke-induced neurogenesis was ameliorated in ER-deficient mice, demonstrating the importance of ER signaling. Fourth, local production of E2 in the brain may contribute to injury-induced neurogenesis, as loss of aromatase led to even more striking deficits in neurogenesis and even greater impairments in behavioral recovery than ovariectomy in WT mice, an effect independent of infarct size. Finally, E2 enhanced behavioral recovery after stroke in both male and female WT mice. Recovery was also slower and less complete in female mice lacking ERα, ERβ, and aromatase, demonstrating an association between reductions in neurogenesis, ER signaling, and behavioral recovery. Given the striking lack of recovery in the knockouts, small reductions in NG may have large, unanticipated effects on behavioral recovery.
It is now well established that neurogenesis occurs throughout adulthood in the mammalian brain, primarily in two areas, the DG of the hippocampus and the SVZ (Altman and Das, 1965
; Lindvall and Kokaia, 2010
). Global and focal ischemic insults induce rapid proliferation within these regions in both animal models and in humans (Nakayama et al, 2010
), and redirect neuroblast migration toward the injured cortex and striatum. These early neurons can form synapses with neighboring cells, although the contribution of these cells to neurologic recovery is not yet known (Hou et al, 2008
). Most studies of postischemic neurogenesis have been performed in male animals, yet the principal mammalian estrogen, E2, has a major role in physiological neurogenesis within the hippocampus. Females in the high estrogen, proestrous state have higher levels of neurogenesis than males (Galea, 2008
; Hojo et al, 2004
), a characteristic that is reversed by ovariectomy and in part by treatment with the nonselective ER antagonist ICI 182,780 (Perez-Martin et al, 2003
). Both ERα
agonists increase cell proliferation in the DG (Mazzucco et al, 2006
). The ERβ
knockout mice show regional neuronal hypocellularity, especially in the cerebral cortex, suggesting that ERβ
has a role in developmental and physiologic neurogenesis (Wang et al, 2003
). Both ERα
are expressed in embryonic and adult rat neural stem cells (Brannvall et al, 2002
; Galea, 2008
; Merchenthaler et al, 2004
) derived from the adult SVZ; however, the role of ERs in physiological NG in vivo
remains a subject of debate.
Our results convincingly show that both ERα
have an important role in stroke-induced neurogenesis. Both receptor subtypes are required, as loss of either ER subtype dramatically abolished ischemia-induced neurogenesis in both the SVZ and DG. This is consistent with recent pharmacological studies demonstrating that administration of a selective ERα
agonist, propyl-pyrazole triol, or ERβ
agonist, diarylpropionitrile, increased cell proliferation in ovx adult female rats (Mazzucco et al, 2006
). However, neither agonist was as effective as E2, consistent with a requirement for engaging both receptor subtypes. It has been well documented that ERα
is upregulated after ischemic injury, and this isoform mediates many of the vascular and acute neuroprotective effects of E2 (Dubal et al, 2001
). We now extend this concept to show that ERα
likely has a major role in injury-induced neurogenesis, but that it does not likely operate alone. Recent work has shown epigenetic modification of the ER after murine stroke; injury induces demethylation of the ER promoter allowing for E2-induced gene transcription (Westberry et al, 2008
). This, in conjunction with the known upregulation of aromatase in injured areas of brain (Garcia-Segura, 2008
; Roselli et al, 2009
), may lead to enhanced E2 signaling, development of both an early protective and later proproliferative environment, with enhancement of growth and survival genes such as BDNF, and increased functional recovery. However, as estrogen levels were not directly manipulated in our knockout studies, caution is warranted that the assumption that estradiol is working via a ligand-dependent effect on the ER has not been directly tested in these studies.
We examined ARKO mice in part to address if endogenous E2 production specifically mediates poststroke neurogenesis, rather than a secondary downstream effect triggered by nonligand activation of the ER. Aromatase is present in the embryonic neocortex, within radial glia, and is upregulated with injury (Garcia-Segura, 2008
; Roselli et al, 2009
). Treatment of rat hippocampal cultures with the aromatase inhibitor, letrozole, decreased the number of proliferative cells and increased apoptosis, an effect that was reversed by E2 (Fester et al, 2006
). This suggests that local central nervous system E2 could contribute to stroke-induced neurogenesis and subsequent recovery. We have previously shown that female ARKO mice have significantly more damage after MCAO than WT ovx mice, demonstrating a role for local brain-synthesized E2 in acute neuroprotection (McCullough et al, 2003
). As ARKO mice had lower levels of neurogenesis than any other strain, including ovx WT females, this suggests that extragonadal synthesis of E2 contributes to stroke-induced neurogenesis. However, these results must be interpreted with caution, as we compared ARKO mice with WT ovx females rather than ARKO littermates. Another caution to be noted is that the ARKO mice had larger infarcts than any other strain. However, the extent of injury did not significantly correlate with the amount of stroke-induced neurogenesis. Our data appear to be consistent with the study by Arvidsson et al (2001)
that showed the amount of stroke-induced neurogenesis, at least in MCAO models, is independent of the amount of cell death. However, the possibility that the extensive ischemic damage in the KO strains led to glial scarring or destruction of the ‘neurogenic niche' remains a possibility.
The downstream mechanism by which E2 acts to enhance neurogenesis is not yet known, but likely involves numerous protective, antiapoptotic, and growth-promoting pathways. One candidate growth factor is BDNF, as several studies have shown that expression of BDNF mRNA and protein is modulated by E2 (Singh et al, 1995
; Sohrabji et al, 1995
). The BDNF promoter is known to have an estrogen response element-like sequence (Sohrabji et al, 1995
). BDNF enhances the number and survival of new neurons derived from the SVZ both in vitro
and in vivo
(Pencea et al, 2001
) under physiological and pathological (Ploughman et al, 2009
) conditions. In this study, BDNF levels were elevated 72
hours after stroke, and were significantly higher in intact females compared with males (). Surprisingly, exogenous E2 administration did not significantly increase BDNF levels over that seen with stroke alone in either ovx females or males. There are several possible explanations for this finding. We only examined one time point after stroke, and either shorter or longer assessments may have led to different results and unmasked effects of E2 on BDNF. Alternatively, it is possible that the dramatic stroke-induced BDNF masked any possible E2 effect, or that injury-induced upregulation of aromatase had already led to significant local production of E2 and a maximal BDNF response. Astrocytes and endothelial cells secrete BDNF and induce neurogenesis in adult neural stem cells (Song et al, 2002
) and may provide a scaffold for migration of newborn cells to injured areas. As our experiments focused on stroke-induced neurogenesis in vivo
, we chose to examine brain homogenates for BDNF; therefore, the specific cell type responsible for stroke-induced increases in BDNF is unclear.
In this study, both male and ovx females pretreated with E2 showed a significant increase in BrdU+/DCX+ colabeling 2 weeks after stroke in the SVZ and DG. By 6 weeks after stroke, most BrdU+ cells developed a mature phenotype, suggesting that these cells mature and survive. Our findings are consistent with those of Suzuki et al (2007)
who documented that pretreatment with physiological doses of E2 enhanced poststroke neurogenesis in the dorsal SVZ of female mice. However, this paper evaluated acute changes (i.e., 96
hours after injury), so functional recovery could not be assessed. As it is well documented that the peak of stroke-induced neurogenesis can occur as late as 3 weeks after injury in rodents (Kuge et al, 2009
), we deliberately avoided early dosing BrdU to avoid uptake by dead or dying cells, or those in the process of repair. Our data show that E2 selectively enhances neurogenesis in the injured brain, as no increase was seen in E2-treated sham mice even after 6 weeks of exposure.
There are several technical caveats in this study. BrdU staining as a means to identify ‘newborn' cells has limitations; especially in models of brain injury such as stroke (Taupin, 2007
). The BrdU incorporation is not invariably a marker of cell proliferation; it is only a marker of DNA synthesis. The BrdU incorporation ordinarily occurs in neurons after cell cycle reentry or after cellular repair, and does not detect the physiological status of the BrdU+ cell. Furthermore, attributing the behavioral benefits seen in E2-treated mice directly to enhanced neurogenesis would be premature. Recent work indicates that ischemia induces newborn neurons that display tetrodotoxin-sensitive Na(+) action potentials and spontaneous excitatory postsynaptic currents by 8 weeks of injury (Hou et al, 2008
), suggesting that some newborn neurons are functional. However, it remains unclear if these neurons successfully integrate into neuronal networks and directly lead to improved recovery. The improvements in our behavioral studies may not be reflective of neurogenesis per se
, presumably because the animals recovered too quickly for improvement to be secondary to ‘repopulation' of damaged circuitry. We speculate that the recovery is more likely secondary to enhanced growth factor signals or local support of damaged, but not irreversibly injured cells that remain in the penumbra. Our BDNF data support this hypothesis. E2 is robustly protective in most preclinical models (McCullough and Hurn, 2003
), and we confirm here that stroke damage was significantly less in both male and female mice treated with E2; however, infarct size was independent of the amount of neurogenesis. Interestingly, all mice, regardless of their treatment status, sex, or genotype had similar behavioral deficits at day 2. This suggests that the behavioral tests used do not reflect initial infarct size but are more sensitive for assessing the rate of behavioral recovery, which was closely related to the amount of poststroke neurogenesis.
The major translational limitation of this work is that animals were treated with E2 before injury. A more relevant approach would be to evaluate the beneficial effects of E2 given poststroke on recovery to avoid possible confounding neuroprotective effects on behavior. Delayed E2 treatment (2 days after stroke) does not improve behavioral recovery in rats, suggesting that the treatment window may be short (Farr et al, 2006
). Poststroke neurogenesis occurs in humans (Nakayama et al, 2010
), but whether enhancing this endogenous response would improve recovery is unclear. Recent studies examining selective ablation of neuronal cell precursors indicate that short-term recovery (24
hours) is linked to the neurogenic response, at least in animals (Jin et al, 2010
). Another limitation of our study is that we did not use older mice. Aging animals exhibit stroke-induced neurogenesis, but this response is ‘muted' compared with young animals (Gao et al, 2009
). As BDNF levels are known to drop with age (Jezierski and Sohrabji, 2001
), this may have major implications if E2-induced neurogenesis is mediated in part by BDNF. Translation to clinical populations could also involve some risk, as the Women's Health Initiative has shown that stroke incidence increased in healthy postmenopausal women treated with E2 although there is evidence that the timing of replacement may be a critical factor (reviewed in Sherwin (2009)
In conclusion, this work shows that E2 contributes to behavioral recovery via enhancement of neurogenesis, suggesting that further work to examine dose and duration of therapy are warranted. As over 80% of the neurons generated after stroke die within the first 2 weeks after their formation (Arvidsson et al, 2002
), strategies to enhance survival and function of these cells are urgently needed. As the incidence of stroke continues to rise in our aging population, despite improved prevention and acute treatments, the mechanisms involved in recovery after stroke and enhancing function will be major goals in the future.