Gonadal steroids prevent cell damage and stimulate behavioral recovery after transient middle cerebral artery occlusion in male and female rats

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Abstract

17β-Estradiol (E) and progesterone (P) are neuroprotective factors in the brain preventing neuronal death under different injury paradigms. Our previous work demonstrates that both steroids compensate neuronal damage and activate distinct neuroprotective strategies such as improving local energy metabolism and abating pro-inflammatory responses. The current study explored steroid hormone-mediated protection from brain damage and restoration of behavioral function after 1 h transient middle cerebral artery occlusion (tMCAO). Male and ovariectomized female rats were studied 24 h after stroke. Both steroid hormones reduced the cortical infarct area in males and females to a similar extent. A maximum effect of ∼60–70% reduction of the infarct size was evident after P and a combined treatment with both hormones. No infarct protection was seen in the basal ganglia. Testing of motor and sensory behavioral revealed an equal high degree of functional recovery in all three hormone groups. Gene expression studies in the delineated penumbra revealed that estrogen receptor (ER) alpha and beta are locally up-regulated. tMCAO-mediated induction of the pro-inflammatory chemokines CCL2, CCL5 and interleukin 6 was attenuated by E and P, whereas the expression of vascular endothelial growth factor (VEGF) was fortified. Local expression of microglia/macrophage/lymphocyte markers, i.e. Iba1, CD68 and CD3, were significantly reduced in the penumbra after hormone treatment suggesting attenuation of microglia and lymphocyte attraction. These results demonstrate the neuroprotective potency of a combined treatment with E and P under ischemic conditions in both sexes and point at the regulation of chemokine-microglia/lymphocyte interactions as a supposable mechanism implicated in cell protection.

Introduction

The steroids 17β-estradiol (E) and progesterone (P) are characterized by their neuroprotective potency in the CNS under acute and degenerative neuropathological challenges. The risk of cardiovascular events in women rises after menopause (Carwile et al., 2009, Roof and Hall, 2000, Wenger et al., 1993) and postmenopausal women are more vulnerable than young women to neurodegenerative diseases (Kipp and Beyer, 2009, Merchenthaler et al., 2003). Sex differences in the incidence rate and the degree/course of pathological brain damage are often associated with disparities in steroid hormone plasma levels between sexes and during lifetime (Acs et al., 2009, Alkayed et al., 1998, Herson et al., 2009, Kipp et al., 2006).

Ischemic neuronal tolerance has been intensively studied in cultured neural cells. There is increasing evidence from animal studies that E and P might limit the ischemic damage following occlusion of the middle cerebral artery (MCA). The administration of physiological levels of E prior, during, or after (days or at least several hours) permanent or transient (t) MCAO leads to a reduction of the infarct volume (Rau et al., 2003, Suzuki et al., 2009, Toung et al., 1998, Zhang et al., 2009). Different cellular mechanisms are debated to be involved in the steroid-mediated dampening of acute brain infarct processes such as diminishing of pro-inflammatory events (Chiappetta et al., 2007), impact on blood circulation and the neurovascular unit by stabilizing endothelial integrity in the CNS (Guo et al., 2010, Suzuki et al., 2009), and oxidative metabolism (recently reviewed by Lebesgue et al., 2009, Sims and Muyderman, 2010). Nonetheless, it appears evident that the attenuation of delayed cell degeneration but not immediate acute cell death may also play a role, and classical nuclear estrogen receptor (ER) signaling appears to be indispensable for this effect (Dubal et al., 2006). This is supported by recent data showing that E substitution in combination with thrombolytic factors may increase the therapeutic window for the treatment of ischemic stroke (Liu et al., 2010a, Liu et al., 2010b). The role of P as a neuroprotectant under ischemic brain conditions is less-defined. It was shown that P enhances functional recovery after MCAO (Gibson and Murphy, 2004, Stein, 2009). Otherwise, P seems to be only effective in co-treatment together with E but not alone (Toung et al., 2004). In general, the route of steroid application, dose-dependent variations, exposure time, duration of infarct, and choice, age and sex of animals are important variables that strongly affect the outcome of neuroprotection in the ischemic brain (Carwile et al., 2009, Draca, 2009, Singh et al., 2008, Strom et al., 2010).

The main objective of the present study was to determine the effectiveness of single vs. combined E and P short-term treatment during 24 h transient tMCAO on the magnitude of the infarct area and the behavioral restoration in male and female rats under defined plasma steroid monitoring. In order to relate neuroprotection to presumable cellular mechanisms and cell–cell interactions, we further used male animals and correlated the neuroprotective magnitude with the expression levels of pro-inflammatory genes and proteins and the attraction/activation of microglia, macrophages, and lymphocytes in the delineated penumbra.

Section snippets

Animals

Male and ovariectomized female Wistar rats (12–14 weeks old, males 380–410 g, and females 270–290 g, Charles-River, Germany) were maintained in a pathogen-free and climate-controlled environment with access to water and food ad libitum handled according to the rules of “Care of Animal Subjects” (North Rhine-Westphalia, Germany). Research and animal care procedures were approved by the Review Board for the Care of Animal Subjects of the district government (North Rhine-Westphalia, Germany).

Results

Laser-Doppler monitoring revealed that in all rats included in the study relative regional blood flow values were reduced by >50% compared to pre-ischemic values within several minutes after induction of tMCAO and remained stable during tMCAO (Fig. 1B). The application of hormones did not influence cerebral blood flow values during the 24 h interval. The overall mortality in the whole experiment was approximately ∼17% and did not show differences between groups.

Fig. 2 depicts the morphological

Discussion

Consistent with earlier results (Carwile et al., 2009, Elzer et al., 2010, Gibson et al., 2009, Toung et al., 2000), we have shown that E and P are individually and in combination neuroprotective after transient focal ischemia and short-term recovery in the cerebral cortex but not in the basal ganglia. Our study demonstrates several important findings. First, the concurrence of both hormones appears to be effective for attenuating cell damage and behavioral restoration which is in accordance to

Conflict of interest statement

All authors declare that they have no conflict of interest.

Acknowledgments

The technical support by H. Helten is acknowledged. The work was supported by IZKF BIOMAT (C.B.) and START (M.K.) of the Faculty of Medicine, RWTH Aachen University as well as B. Braun Melsungen AG (Germany).

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