Research ReportFailure of estradiol to improve spontaneous or rehabilitation-facilitated recovery after hemorrhagic stroke in rats
Introduction
The incidence of stroke is lower in pre-menopausal women than in men, which has been largely attributed to the high levels of estrogen, predominantly 17β-estradiol (E2), circulating in women (Ayala et al., 1995, Bushnell et al., 2006, Prentice, 2007, Wise et al., 2005). Unexpectedly, the Women's Health Initiative (WHI) trial reported that estrogen replacement therapy actually increased stroke incidence in postmenopausal women (Wassertheil-Smoller et al., 2003); although it has been argued that this trial has serious design flaws (Bushnell et al., 2006, Klaiber et al., 2005, Wise et al., 2005) Regardless, the potential of using E2 to protect the brain (neuroprotection) in both women and men following stroke is of great interest. Indeed, in experimental models of global cerebral ischemia E2 treatment has been repeatedly shown to attenuate hippocampal CA1 sector injury (Jover et al., 2002, Shughrue and Merchenthaler, 2003, Sudo et al., 1997). Similarly, E2 reduces infarct size following focal ischemia (Dubal et al., 1998, McCullough et al., 2001, Simpkins et al., 1997). It is not surprising then that E2 pretreatment reduces injury in models of intracerebral hemorrhage (ICH) that target the striatum (Auriat et al., 2005, Nakamura et al., 2005). Estrogen is thought to exert its neuroprotective actions against ischemia via numerous mechanisms (Gibson et al., 2006). Direct mechanisms include preserving cerebral blood flow and enhancing post-ischemic reperfusion (Hurn and Brass, 2003) as well as suppression of pro-apoptotic signals (Dubal et al., 1999, Harms et al., 2001). While similar mechanisms likely contribute to E2's neuroprotective effects in ICH models, estrogen may further affect ICH by promoting hemostasis and lessening the hematoma size following vessel rupture (Auriat et al., 2005). While ischemia and ICH share many mechanisms of injury, fundamental differences remain (Xi et al., 2006). Thus, it is important to test putative treatments in ICH models rather than rely on findings in ischemia models.
While the ability of E2 to reduce injury, such as CA1 sector cell loss after global ischemia, has translated into improved functional recovery (e.g., memory tasks) (Auriat et al., 2005, Gulinello et al., 2006, Li et al., 2004a, Li et al., 2004b), it is also well known that E2 affects brain function in ways that should independently improve functional recovery. For example, E2 promotes the formation of new dendrites and excitatory synapses in the hippocampus (Woolley and McEwen, 1992), and this effect is correlated with improvements in hippocampal-dependent memory (McEwen and Woolley, 1994, Packard and Teather, 1997). As well, E2 affects the expression of brain-derived neurotrophic factor (BDNF) (Berchtold et al., 2001, Sohrabji et al., 1995), which not only promotes neuronal survival but also regulates plasticity (Alderson et al., 1990, Lindsay, 1988). Interestingly, E2 receptors in the forebrain are co-localized with BDNF (Miranda et al., 1993), and E2 replacement in ovariectomized (OVX) female rats increases BDNF expression in the forebrain (Allen and McCarson, 2005, Jezierski and Sohrabji, 2000). Accordingly, E2 may affect recovery by enhancing dendritic arborization and spine density (Berchtold et al., 2001, Jezierski and Sohrabji, 2003, McEwen, 2001), among other mechanisms, and these may be used to improve spontaneous recovery after stroke. Effective rehabilitation therapies also promote increases in dendritic arborization and spine density following stroke (Biernaskie and Corbett, 2001). Thus, it is possible that E2 treatment may further enhance rehabilitation efforts.
In this study, we assessed whether delayed E2 treatment affects spontaneous and rehabilitation-facilitated recovery after ICH in rats (Fig. 1—timeline of events). The collagenase model of ICH (Rosenberg et al., 1993) was used because it produces relatively consistent bleeding within the striatum along with well-characterized behavioral deficits (Auriat et al., 2008, MacLellan et al., 2006). Beginning 1 week after ICH, rats were group housed in either standard (STD) or EE cages and received either E2 pellets or they underwent sham procedures (SH). We used environmental enrichment (EE) as a rehabilitation treatment as it improves functional recovery following several types of brain injury (for a review, see Will et al., 2004), including ICH in rats (Auriat et al., 2008). Rats were group housed in the EE cages, which contained ramps, tunnels, beams and various objects to explore. Estrogen was administered continuously, via an implanted pellet, until the end of the study to mimic estrogen replacement therapy. Furthermore, the onset of E2 treatment was delayed to 1 week post-ICH to isolate E2's ability to independently promote functional recovery from its direct effects on cell death and hematoma size found with pretreatment (Auriat et al., 2005). Comprehensive histological and functional evaluation at long survival times has been highly recommended in the assessment of putative stroke therapies (Recommendations for standards regarding preclinical neuroprotective and restorative drug development, 1999, MacLellan et al., 2006). Thus, we gauged recovery at 2, 5 and 8 weeks after ICH with several well-characterized behavioral tests that included: the tray task to assess skilled reaching (Whishaw et al., 1986), the cylinder test of forelimb use asymmetry (MacLellan et al., 2006, Shanina et al., 2006), the horizontal ladder walking test (MacLellan et al., 2006, Metz and Whishaw, 2002) and the elevated beam task to assess balance and locomotion (Feeney et al., 1982, MacLellan et al., 2006). We predicted that delayed EE would promote functional recovery and that delayed E2 would facilitate recovery independently of and in combination with EE. Neither treatment was expected to alter lesion size.
Section snippets
Protocol violations and mortality
Thirteen rats were excluded entirely from this study. Of these, 7 died during surgery of unknown causes, likely due to anesthetic complications. Six others were excluded due to surgical errors (e.g., no lesion likely due to a blocked injection needle). The remaining group sizes were as follows: STD-SH (n = 16), STD-E2 (n = 12), EE-SH (n = 14), and EE-E2 (n = 17). Some data for several other rats were excluded from one or more behavioral tests for failing to meet criteria stated in Experimental
Discussion
This is the first study to assess whether delayed and chronic E2 treatment influences functional recovery after striatal ICH in rats. A sustained E2 treatment beginning 1 week following collagenase-induced ICH did not notably influence functional recovery or brain injury. Environmental enrichment did facilitate recovery on some tests, but this was mostly unaltered by E2 treatment. These findings, in conjunction with previous work (Auriat et al., 2005, Li et al., 2004b), suggest that E2
Animals
Seventy-two female Sprague–Dawley rats were entered into this study. They were obtained from the Biosciences breeding colony at the University of Alberta and were cared for in accordance with the Canadian Council on Animal Care guidelines. Procedures were also approved by the Biosciences Animal Care and Use Committee at the University of Alberta.
Rats were housed in groups of 3 to 4 in standard polycarbonate cages (38 cm width × 49 cm length × 20 cm height) unless otherwise stated, and on a 12 h
Acknowledgments
Research was supported by grants from the Canadian Stroke Network and the Canadian Institutes of Health Research to FC who is supported by an Alberta Heritage Foundation for Medical Research senior medical scholar award. APN was supported by a Natural Sciences and Engineering Research Council Masters Post Graduate Scholarship. The authors gratefully acknowledge T. McInnes, A. Black, A. Yushchyshyn and M. Fingas for their technical assistance. C. MacLellan kindly provided helpful comments on the
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Cerebrovascular Stroke: Sex Differences and the Impact of Estrogens. Sex Differences and the Impact of Estrogens.
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2014, Behavioural Brain ResearchCitation Excerpt :In addition, treatment with the ERβ ligand diarylpropionitrile enhanced functional remyelination in experimental autoimmune encephalomyelitis which also is of importance for mechanisms of recovery in the postischemic brain [29]. In contrast, other studies using male as well as female rats failed to achieve beneficial effects of estrogen on recovery following permanent occlusion of the middle cerebral artery or intracerebral haemorrhage [30–32]. The nonsteroidal selective ERβ agonist 4-(1-phenyl-cyclohexyl)-phenol (AC-131) has been shown efficacy in the reduction of pain in animal models involving nerve injury and chronic inflammatory pain [33,34] at dosages where no classical estrogenic functions mediated by activation of ERα were observed in vivo [33].
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2009, Brain ResearchCitation Excerpt :This is the first collagenase ICH report of forelimb placement and forelimb use asymmetry this long after injury. The incomplete recovery of forelimb placement is an important finding for future therapeutic applications, whereas the asymmetry result should direct future studies to assess time points at 8 weeks (Nguyen et al., 2008) or earlier. The collagenase model of ICH produced detectable cognitive and motor deficits in adult rats that evolved over a 10 week time period, and provides a suitable baseline for the analysis of therapeutic intervention strategies following stroke.
Delayed rehabilitation lessens brain injury and improves recovery after intracerebral hemorrhage in rats
2009, Brain ResearchCitation Excerpt :Alternatively, it is possible that our REHAB treatment did not provide enough walking practice to persistently improve outcome. Indeed, EE treatment alone when given all day for a longer period does diminish stepping errors after ICH (Auriat and Colbourne, 2008; Nguyen et al., 2008a). Use of EE alone does not lessen limb use asymmetry in the cylinder task after striatal ICH (Nguyen et al., 2008a).
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2008, Experimental Neurology