In the present study, we found that administration of 17β-estradiol at the onset of ischemia diminished the deleterious effects of tPA on neurobehavioral outcome. In addition, we observed that E2 countered another major side effect of tPA, i.e., its augmentation of ischemic-induced damage to the BBB. By maintaining a functional BBB, E2 inhibited tPA-associated intraparenchymal hemorrhage. The latter advantage may largely result from E2’s suppression of proteolysis by MMP-9. These benefits were independent of changes in core temperature, CBF, and other physiologic parameters, none of which differed much among the control and treated groups.
When tPA is administered shortly after the onset of ischemia, it stimulates thrombolysis. As this process restores blood flow, it can reduce the loss of brain tissue. In some cases, tPA-treated stroke victims have recovered without any symptoms (Alexandrov et al., 2004
). Administration of tPA in the aftermath of ischemic stroke, however, increases the risk of hemorrhagic transformation and neurotoxicity, which may aggravate the outcome of ischemic stroke (Audebert et al., 2006
; Cocho et al., 2005
Estrogen is known to be neuroprotective in experimental ischemic stroke (Dubal and Wise, 2001
; Fan et al., 2003
; Liu et al., 2005
; Rusa et al., 1999
; Toung et al., 1998
; Yang et al., 2000
) and is known to preserve BBB integrity with consequent reduction in brain edema (Crosby et al., 2007
; O’Donnell et al., 2006
; Rusa et al., 1999
; Saleh et al., 2001a
; Toung et al., 1998
). However, hormone replacement therapy was not found to be beneficial for reducing clinical stroke outcome (Wassertheil-Smoller et al., 2003
). Nevertheless, acute administration of a large dose of estrogen may be beneficial. Pretreatment with E2 has been reported to counter disruption of the BBB and decrease brain edema in transient focal cerebral ischemia without tPA (Liu et al., 2005
). Recently, combined use of estrogen with tPA has been reported to significantly reduce infarct volume after 1 h of MCAO (Liu et al., 2010
). The present study adds to our knowledge of the potential for acute estrogen therapy by demonstrating that estrogen ameliorates the adverse effects of tPA on MMP-9 activation, EB extravasation, brain edema, tissue hemoglobin content, and neurological deficits after 2 h of MCAO. Thus to our knowledge, the present study is the first to report that E2 counters tPA’s effects of weakening the BBB and augmenting hemorrhagic transformation.
The disruption of the BBB is a complex process, one thought to be orchestrated by several molecular mechanisms. Among the most important is the activation of MMPs (Sumii and Lo, 2002
; Tsuji et al., 2005
). Once activated, MMPs degrade vascular basal lamina and other constituent structures of the BBB with consequent edema and vascular rupture (Kelly et al., 2006
; Lo et al., 2004
). Studies in knockout mice have identified MMP-9 to be one of the principal MMPs activated after ischemia (Kelly et al., 2006
; Tsuji et al., 2005
). Moreover, administration of tPA following ischemic stroke was found to augment the activation of MMPs. An equivalent phenomenon has been observed in clinical practice: patients who suffer hemorrhagic conversion following treatment with tPA have unusually high levels of plasma MMP-9 activity (Montaner et al., 2003
). In vitro studies indicate that MMP-9 is upregulated in cerebral microvascular endothelial cells by tPA (Wang et al., 2003
), and that estrogen reduces cerebrovascular inflammation(Razmara et al., 2005
) and protects endothelial cells from stress (Guo et al., 2010
; Razandi et al., 2000
; Razmara et al., 2008
). MMP-9 is thought to be the key molecule in tPA induced injury. In the present study, we also found that tPA-treatment of ischemic rats increased activation of MMP-9, whereas tPA did not augment the increase in MMP-2 activity. These findings are in agreement with others (Tsuji et al., 2005
). Moreover, we found that co-administering E2 caused a significant blunting of MMP-9 activity following MCAO with tPA treatment. These results suggest that E2 diminishes the damage to the BBB incurred by tPA by suppressing MMP-9 activity.
The adverse effects of tPA in stroke are augmented when tPA administration is delayed. In our protocol, E2 was administered subcutaneously at the onset of MCAO, and MCAO persisted for 2 h before administering tPA at reperfusion. Although we observed a trend for a reduction in infarct volume with E2 treatment, the difference was not significant. A recent paper reported that E2 treatment 2 h before MCAO decreased infarct volume after 1 h of MCAO with tPA treatment at reperfusion (Liu et al., 2010
). However, in that study tPA treatment alone at 1 h did not augment infarct volume, whereas we observed a significant augmentation of infarct volume when tPA was administered alone at 2 h. Together, these findings suggest that the reduction in infarct volume with acute treatment of E2 is more robust with a shorter ischemic duration when the severity of the injury is less severe and the adverse effects of tPA are less prominent. Alternatively, the 2-hour pretreatment used by Liu et al. (2010)
may have allowed more time to affect changes in the neurovascular unit before the onset of ischemia. Therefore, the duration of MCAO and the timing of E2 and tPA administration are likely important in determining the impact of treatment on infarct volume (Yang et al., 2000
). Further work is required to define the therapeutic window for E2 administration, whether E2 would be efficacious when administered after tPA, and whether E2 efficacy would be lost when tPA administration is delayed beyond 2 h and BBB damage is further augmented. Nevertheless, the present study together with the work of Liu et al. (2010)
provides a proof-of-concept that E2 can be added to the list of agents that can protect the neurovascular unit from the adverse effects of tPA.
In conclusion, we found that administration of 17β-estradiol reduces tPA-induced augmentation of MMP-9 activity, BBB permeability, cerebral edema, cerebral hemorrhage and neurological deficits after reperfusion from 2 h of MCAO in rats. Thus, acute administration of E2 has the potential to serve as a prophylactic therapy against tPA’s life-threatening side effect of cerebral hemorrhage.