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

Brain, Behavior, and Immunity 25 (2011) 715–726

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Gonadal steroids prevent cell damage and stimulate behavioral recovery after transient middle cerebral artery occlusion in male and female rats Jon Dang, Bhimashankar Mitkari, Markus Kipp 1, Cordian Beyer ⇑,1 Institute of Neuroanatomy, Faculty of Medicine, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany

a r t i c l e

i n f o

Article history: Received 27 October 2010 Received in revised form 12 January 2011 Accepted 21 January 2011 Available online 28 January 2011 Keywords: Stroke MCAO Estrogen Progesterone Microglia Chemokine

a b s t r a c t 17b-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. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction The steroids 17b-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 ⇑ Corresponding author. Fax: +49 (0) 241 80 82 472. 1

E-mail address: [email protected] (C. Beyer). Equal senior co-authorships.

0889-1591/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2011.01.013

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,b). 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

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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. 2. Materials and methods 2.1. 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). Ovariectomy was performed by the animal provider approximately 2–3 weeks before tMCAO. A total of 63 animals (51 males and 12 females) were tested in this study. Eight males and three females died during MCAO recovery. The time-course from animal surgery until sacrificing including all steps of treatment and analysis is schematically presented in Fig. 1A. 2.2. Surgery and tMCAO Anesthesia was induced by inhalation of 5% isoflurane (Abbott, Ludwigshafen, Germany) for 2 min and maintained at 2–2.5% (depending on each animal and operation step) using a face mask. Body temperature was kept throughout surgery at 37 ± 0.5 °C using

a heating blanket and lamp. Laser-Doppler flowmetry (Moor Instruments VMS-LDF2, Axminster, UK) was used to monitor cerebral blood flow on the ipsi- and contralateral side for 1 h during surgery (Fig. 1B). The 2 mm Laser-Doppler measuring sensors were placed 1–2 mm posterior and 4–5 mm lateral to the bregma on the left and right skull hemisphere depending on highest blood perfusion units (BPU) after a small midline skin incision at the ipsilateral side. To ensure that the BPU digitized the cortical vascular territory of the middle cerebra artery (MCA), the common carotid artery was clipped temporary resulting in a decrease of BPU. Prior to fixation of the sensors, the skull was carefully thinned out to assure that the reduction of BPU is not due to reduction in external carotid artery (ECA) blood flow. Focal cerebral ischemia was induced by occlusion of the left MCA as previously described (Herrmann et al., 2005; Kramer et al., 2010) with some minor modifications. Following a small midline neck skin incision, the left common carotid artery (CCA), internal carotid artery (ICA), and ECA were exposed. Subsequently, the ECA was transiently clipped and the CCA was ligated proximal. The vagus nerve was carefully preserved. Directly before insertion of the catheter into the ICA, baseline values of cerebral blood flow (CBF) given as BPU were measured. To avoid protrusion into the A. pterygopalatina, the head was bended aside and a commercially available catheter (Asahi PTCA Guide Wire Soft, Abbott Vascular) was subsequently introduced from the lumen of the distal CCA at the bifurcation until an immediate drop in baseline CBF occurred. Only those animals were included which showed a reduction of regional CBF by >50% compared to baseline before tMCAO.

2.3. Hormone treatment and analysis All steroid hormones were from Sigma–Aldrich, Munich, Germany. Rats subjected to tMCAO were randomly assigned to receive E, P, E/P, or vehicle (sesame oil plus ethanol as solvent) treatment. Steroids were initially dissolved in ethanol and further diluted in sesame oil to obtain final steroid concentrations in the experiment. The following steroid concentrations were routinely used: E (25 lg/kg body weight) and P (10 mg/kg body weight). For studying dose-dependency, the following concentrations were applied: E (2.5 lg/kg and 0.25 lg/kg body weight) and P (1 mg/kg and 0.1 mg/kg body weight). Steroids were subcutaneously applied

Fig. 1. Schematic illustration of the experimental procedure of tMCAO and hormone substitution (A). Representative Laser-Doppler measurement (n = 13 males, 9 females) of relative cerebral blood flow in the ipsilateral middle cerebral artery territory before and after tMCAO until sacrificing animals after 1 h (B). Relative CBF was reduced by >50% in both sexes and not affected by hormonal treatment. BS, blood sampling; BT, behavioral testing; CBF, cerebral blood flow; H1, first hormone application; H2, second hormone application; S, sacrificing; SUR, surgery; TP, tissue processing.

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as neck depots (500 ll) immediately after the withdrawing of the catheter and 12 h later. This procedure and the chosen concentrations were found in preliminary dose-finding studies to yield stable plasma hormone levels corresponding to typical female estrous hormone levels already after 4 h of application and throughout the 24 h recovery period (not shown). After behavioral testing, blood samples were taken from every tested animal by cardiac puncture to determine the individual steroid hormone plasma levels. Control animals (i.e. sham-operated; the catheter was not inserted in the ICA) underwent the same experimental protocol, except that they received the same vehicle volume only. Total plasma hormone titers of P and E were commercially analyzed by Dialog Service GmbH (Frankfurt am Main, Germany) using a standardized electrochemical luminescence technique and commercially available kits (Roche Diagnostic GmbH, Mannheim, Germany with detection limits of 5.0 pg/ml for E and 30 pg/ml for P). 2.4. Behavioral testing We assessed post-ischemic neurological deficits using motor and sensory behavioral tests with minor modifications as described (Garcia et al., 1995). Testing was performed prior sacrificing by two blinded readers. The following six tests were performed. Spontaneous activity was analyzed for 3 min within an unfamiliar environment by placing the animals in the middle of a 35 cm  55 cm sized cage (scores: 3 = rat moving around, exploring the environment, and approaching at least three walls of the cage; 2 = slightly affected rat moving around in the cage but not approaching the walls and hesitating to move further, nonetheless eventually rising to at least one upper rim of the cage; 1 = severely affected rat not rising up at all and barely moving in the cage; 0 = rat not moving). For testing forepaw outstretching, rats were fixed at the tail, and the symmetry of the outstretching of both forelimbs was evaluated (scores: 3 = both forelimbs outstretched symmetrically; 2 = right side moves and outstretches less than left side; 1 = right side moves slightly; and 0 = right forelimb not moving). To assess ability to climb, rats were placed on the wall of a wire cage. Normally, rats use all four limbs to climb the wall (scores: 3 = rat climbing easily and gripping the wire tightly; 2 = right side impaired when climbing or not gripping as tightly as the left side; and 1 = rat failing to climb or tending to circle instead of climbing). To test body proprioception, rats were touched with a blunt stick on each side of the body, and the reaction to the stimulus was evaluated (scores: 3 = rat reacts by turning head and being equally startled by the stimulus on both sides; 2 = rat reacts slowly to stimulus on right side; and 1 = rat not responding to stimulus placed on the right side). Spontaneous walking activity was staged (scores: 3 = rat walking straight ahead; 2 = right circling; 1 = rat tending to walk toward the right side; and 0 = rat not moving). Sensory function was tested by brushing the vibrissae (scores: 3 = rat turns head to the stimulus side; 2 = rat reacts slowly to stimulus on right side; and 1 = rat did not respond to stimulus on right side). Individual scores of all tests were summed. An overall minimum score of 3 and maximum score of 18 points was achievable. 2.5. Tissue staining and infarct volume At 24 h after tMCAO, all animals were deeply anesthetized again (5% isoflurane) and received an intraperitoneal injection of an overdose of pentobarbital (328 mg/kg). Afterwards, animals were transcardially perfused with physiological NaCl. Brains were dissected and placed in a rat brain matrix (Alto Brain Matrix stainless steel 1 mm rat coronal 300–600 g; Harvard Apparatus, Holliston, MA,

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USA). Two millimeter coronal slices were cut and incubated for 20 min at 37 °C in a 2% (w/v) 2,3,5-triphenyl-tetrazoliumchloride solution (TTC, Fluka, Germany) solved in physiological NaCl for determination of the infarct size. TTC is a marker for metabolic function and represents a reliable indicator of ischemic areas for up to 3 days after ischemia (Benedek et al., 2006; Kramer et al., 2010; Lin et al., 1993) Metabolic active tissue is typically stained red after TTC exposure, whereas necrotic tissue remains pale. Coronal slices were arranged in a frontal to occipital order, and digital photographs of all stained slices were taken using a Canon Digital IXUS 9015 camera (Canon, Tokyo, Japan). Areas of ischemic damage were evaluated in a blinded manner using the open access morphometric software ImageJ 1.41 (NIH, Bethesda, MD, USA). Total infarct volumes were calculated by adding the mean-area of each section and multiplied by 2 mm (thickness of the sections). Edema correction of the infarct volume was performed using following paradigm: volume correction (infarct volume  contralateral volume/ipsilateral volume) (Swanson et al., 1990). The infarct volume was separately calculated for the cerebral cortex (Cx) and the basal ganglia (BG). For immunohistochemistry, rats were transcardially perfused with 2% (w/v) paraformaldehyde (Roth, Germany) containing 15% (v/v) saturated picric acid at pH 7.4 (AppliedChem GmbH, Germany). Brains were post fixed overnight in the same fixative and paraffin-embedded the next day. Then, brains were paraffinembedded (Merck KGaA, Germany). Thereafter, coronally sections each 5 lm thick were made and rehydrated using standard protocols. Slices were incubated with blocking serum for 1 h at room temperature, following exposure to primary antibodies overnight (anti-NeuN (1:500, Abcam, Cambridge, UK), anti-ERa (1:250, Abcam, Cambridge, UK), and anti-ionized calcium-binding adaptor molecule 1 (Iba1, 1:250, Wako, Osaka, Japan). After several washing steps, sections were incubated with biotin-conjugated secondary antibody for 1 h, and subsequently with a biotin– avidin–enzyme complex (Vectastain ABC kit, Vector Laboratories, Burlingame, USA). Antibody binding was visualized by substrate incubation (AEC Substrate kit, Invitrogen, Camarillo, USA). For quantification of Iba1-positive cells and ramification analysis, six slices between the anterior commissure and the posterior commissure were analyzed. 2.6. RNA isolation and real-time PCR Gene expression studies were solely performed with tissues corresponding to the penumbra region (see Fig. 2). Cerebral cortical tissue from TTC-stained coronal brain sections were prepared, the penumbra region dissected using a stereomicroscopic approach, dissolved in lysis buffer (NucleoSpinÒ RNA/Protein kit, Machery-Nagel, Germany), and homogenized with 1.4 mm diameter ceramic beads (Precellys 24, Peqlab, Germany) at 5000 rpm for 15 s. RNA and protein were subsequently isolated from this penumbra tissue following a recently published protocol (Kramer et al., 2010). Purity was controlled using 260:280 OD ratios (NanoDrop 1000, Peqlab, Germany). Reverse transcription reactions were performed using the MMLV RT-kit and random hexanucleotide primers (all from Invitrogen, Germany). Real-time rtPCRs (RT-rtPCRs) were carried out in a mixture consisting of 2 ll cDNA, 2 ll RNAse-free water (Invitrogen, Germany), 5 ll 2xSensi Mix x Plus SYBR & Fluorescein (Quantace), and 0.5 ll primers (10 pmol/ll). Reactions were conducted in standard tubes using the MyIQ RT-rtPCR detection system (Bio-Rad, Germany) under following conditions: 10 min enzyme activation at 95 °C, 40 cycles of 15 s denaturation at 95 °C, 30 s annealing at individual temperatures, 30 s amplification at 72 °C, and 5 s fluorescence measurement at 72 °C. External standard curves were generated by several fold dilutions of the target genes. The concentration

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Fig. 2. TTC-stained 2 mm coronal brain slice of a vehicle-treated male rat shows the white infarct area 24 h after tMCAO. Red color indicates intact tissue (A). NeuN-staining of a consecutive 5 lm coronal brain slice of a vehicle-treated male rat reveals a more precise morphological description of vital and dying/dead neurons and allows defining the penumbra region. Note the presence of mainly dystrophic cells and parenchymal vacuoles in the core region of the infarct area (C and D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of the target genes was calculated by comparing Ct values in each sample with Ct values of the internal standard curve as described previously (Acs et al., 2009; Kramer et al., 2010). Values were normalized using expression values of the housekeeping genes (hypoxanthin–guanin–phosphoribosyltransferase) HPRT and cyclophilin A. PCRs were performed in duplicate. Melting curves and electrophoretical analysis of the RT-rtPCR products (10 ll) plus ethidium bromide staining were routinely performed to specificity RT-rtPCR. For illustration in graphs, expression values from the ipsilateral side of placebo-treated MCAO animals were set to 100% and the corresponding data from hormone groups were always shown as percentage of this normalized value. A list of all primers (Invitrogen, Germany) designed in the free-access internet program ‘‘Primer 3’’ is given (Table 1).

Table 1 List of primer used for real-time PCR. Primer

Sequence

Length (bp)

ERa

s 50 -CCAAAGCCTCGGGAATGG-30 as 50 -AGCTGCGGGCGATTGAG-30 s 50 -TGCTGGATGGAGGTGCTAATG-30 as 50 -CGAGGTCGGGAGCGAAA-30 s 50 -AATGTGTCCTTCCCACAAGC-30 as 50 -GGCAGCAAGAGAGATTGGTC-30 s 50 -AGCTCCCAGACGAAAAGACA-30 as 50 -GAGGGCTGGTTTGGCTTT-30 s 50 -GGTCCATTCCTATGACTGTAGATTTT-30 as 50 -CAATCAAGACGTTCTTTCCAGTT-30 s 50 -GGCAAATGCTGGACCAAACAC-30 as 50 -TTAGAGTTGTCCACAGTCGGAGATG-30 s 50 -AGAACTGCATGGAGGTGGAC-30 as 50 -TTTCGGATGGGCTCATAGTC-30 s 50 -ACAGTGCATCATCGCTGTTC-30 as 50 -CCGGAGAGGAGACTTCACAG-30 s 50 -GGCATGCTGGACCCAAGCTC-30 as 50 -GCGCTTGCTCCTGTGAGTCC-30 s 50 -CACATAGGAGAGATGAGCTTC-30 as 50 -CCGCCTTGGCTTGTCACAT-30 s 50 -CACATAGGAGAGATGAGCTTC-30 as 50 -CCGCCTTGGCTTGTCACAT-30 s 50 -CTGTTACCTGCTCAGCACCA-30 as 50 -AAAGGCTGCTGGTCTCAAAA-30 s 50 -TGCCCACGTGAAGGAGTATTTTTA-30 as 50 -TGGCGGTTCCTTCGAGTGACAA-30

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ERb CD68 PR HPRT Cyclophilin A CD3 Interleukin 6

2.7. Western blotting and ELISA Levels of VEGF, CCL2 and CCL5 within the penumbra region were quantified by commercially available ELISA kits according to the manufacturer’s instructions (all Peprotech, Germany). CD3 and IL6 protein levels were detected by Western blotting. For ELISA assays, penumbra tissues were homogenized with 1.4 mm diameter ceramic beads as described above in 1 ml PBS (pH 7.4) containing a cocktail of protease inhibitors (Mini, Roche Diagnostics GmbH, Germany). Cell suspension was centrifuged for 5 min at 2000 rpm and supernatant recovered. Protein levels were normalized to entire protein content determined by BCA assay (Pierce, USA). ELISA measurements were performed by quantifying the absorbance at 450 nm with corrections for 540 nm using a microplate reader (Bio-Rad, Germany). For calibration, standard concentrations of the corresponding proteins were routinely included. For Western blotting, the following primary antibodies were used: anti-b-actin, Sigma, USA, 1:1000, 42 kDa, anti-CD3, Abcam, Cambridge, UK, 1:100, 16 kDa, anti-IL6, Abcam, Cambridge, UK, 1:100, 22 kDa. Band intensities were determined with AlphaEaseFC™ software V 4.0.0 (Alpha Innotech, CA, USA). Briefly, after precipitation, proteins were washed with 95% ethanol instead of 50% to remove TTC-staining products. After that, proteins were

NGF VEGF-A CCL2 CCL3 CCL5

81 232 191 125 195 231 160 460 227 191 187 80

s, sense; as, anti-sense; bp, base pairs.

exposed to reducing agents (NucleoSpinÒ RNA/Protein kit, Machery-Nagel, Germany) for SDS–gel electrophoresis. Samples were mixed with Protein Solving Buffer and subsequently incubated for 30 min with Quantification Reagent (both constituents of the kit). Quantitative analysis of Western blots was densitometrically accomplished with a fluorescent scanner (ImageMaster, Pharmacia, Germany) as described (Kramer et al., 2010). Total protein concentrations were determined in 20 ll aliquots with bichinonic acid (Pierce Biotechnology Inc., Rockford, IL, USA) at 37 °C against a standard dilution series of bovine serum albumin. Forty microgram proteins were loaded per lane on a 14% SDS– PAGE and electrophoretically separated and then blotted (15 V,

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Fig. 3. Hormone plasma values in male and ovariectomized female tMCAO rats 24 h after the beginning of ischemia under control tMCAO (no steroid substitution) and MCAO plus steroid treatment immediately and after 12 h tMCAO initiation. Blood samples were taken by heart puncture. Note that dose-dependent application and subsequent plasma analysis were only performed in the male group. Data represent means ± SD.

Fig. 4. Infarction area and volume measurement in male and female ovariectomized rats after 24 h ischemia. In (A), representative TTC-stained slices of a male rat are shown demonstrating the hormone-dependent and hormone-specific reduction of the lesion volume. Panels (B) and (C) show sets of consecutive rostral–caudal TTC-stained slices of male rats revealing protection by E/P at different brain coordinates. Quantitative data of the measurement of lesion volumes are given in (D) (cerebral cortex, male), (E) (cerebral cortex; male, dark bars and female, light-colored bars), and (F) (basal ganglia, male). No clear-cut dose–response was seen when using lower E and P concentrations (dotted bars in (D)). Hormone levels in (E) and (F) correspond to those shown in (D) (black bars). E, 17b-estradiol; n, number of tested individuals per group; P, progesterone; ⁄ P 6 0.01 hormone vs. tMCAO in male; ⁄⁄P 6 0.001 hormone vs. tMCAO in male; ⁄⁄⁄P 6 0.05 male tMCAO vs. female tMCAO. Data represent means ± SD.

45 min, Bio-Rad, Germany) to a polyvinylidene difluoride (PVDF) membrane (45 lm pore size; Roche, Germany). Unspecific binding was prevented by blocking with 5% dry milk in TBS-T for 30 min. Blots were incubated with primary antibodies diluted in 5% milk TBS-T overnight. After washing and incubating with horseradish peroxidise-conjugated secondary antibodies, labeled proteins were visualized with ECL-reagent (GE Healthcare, Amersham, UK). Specific binding was detected with the enhanced chemo-luminescence (ECL) detection system (GE Healthcare, UK) on autoradiography films (Eastman Kodak, Rochester, NY, USA). Band intensities were measured with ImageJ 1.41 (NIH, Bethesda, MD, USA).

2.8. Data analysis All data are given as arithmetic means ± SD/SEM (specified in the figure legends). If not otherwise stated, multiple comparisons (infarct size, brain edema, body weight, neurological deficit, gene expression, microglia number and morphology, protein values) were evaluated by one way ANOVA (analysis of variance) and further evaluated by non-parametric Mann–Whitney U-test or an independent sample t-test with SPSS software version 17.0 (SPSS Inc., Chicago, IL, USA and Graph Prism). The criterion for statistical significance was set at P 6 0.05.

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Fig. 5. Behavioral testing of male and ovariectomized female rats 24 h after tMCAO and hormone substitution (25 lg E/10 mg P). A total of six tests were performed resulting in a maximum of 18 scores in sham-operated animals (no tMCAO, no hormone). Panel (A) summarizes the data of all six scorings. Panel (B) shows the individual scores of the six tests in male animals. E, 17b-estradiol; n, number of tested individuals per group; P, progesterone; ⁄P 6 0.001 tMCAO vs. sham; ⁄⁄P 6 0.01 tMCAO vs. tMCAO plus E, P, or E/ P. Data represent means ± SEM and are given in arbitrary units.

3. 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 appearance of the infarcted brain in a representative brain slice in a vehicle-treated rat by comparing conventional TTC-staining and anti-NeuN-staining. The lesion is confined to the ipsilateral striatal region and the anterior and posterior cerebral cortical vascular MCA territories as indicated by the unstained area (Fig. 2A). This morphological picture of the infarct area is affirmed by NeuN-staining which labels neurons and allows a marked delineation of the healthy and infarcted area, i.e. the penumbra (Fig. 2B). Higher magnifications detail different aspects of neuronal morphology between healthy and damaged regions in the cerebral cortex, i.e. tissue shrinkage, nuclear condensation, and vacuoles. Endogenous steroid plasma levels were analyzed in male and ovariectomized female rats immediately before sacrificing. In males, placebo animals showed low E and P values compared to ovariectomized females which exhibited low but still higher hormone levels compared to males (Fig. 3). Substitution with high doses of E or P or E/P yielded in both sexes similar plasma hormone levels. These established doses were further used in all following protection studies. The application of lower E/P doses to males resulted in much lower E/P plasma concentrations not differing significantly from un-

treated controls. The lower E/P treatment doses revealed no or only moderate protection from MCAO in males (see below, Fig. 4D). No dose-dependent studies with females were performed. Treatment with steroid hormones significantly reduced the infarct area in the cerebral cortex but not in the basal ganglia (Fig. 4). The rostral–caudal spread of tissue damage precisely corresponds to the vascular territory of the MCA (Fig. 4B). After hormone supplementation, lesion volumes were decreased by 37% (E), 68% (P), and 65% (E/P) in the male cerebral cortex (Fig. 4D). The co-application of E and P at lower steroid concentrations showed a tendency but no clear-cut dose responsiveness in the reduction of the lesion volume in male rats (Fig. 4D). In ovariectomized females, the infarct area volume after tMCAO without hormone treatment was significantly lower (17.4 mm3) compared to males (25.8 mm3) (Fig. 4E). The application of E/P yielded similar protective effects as observed in males with a reduction of the infarct volume by 55%. Behavioral testing of male and ovariectomized female rats 23 h after tMCAO and hormones substitution showed that all three combinations of hormones were effective in improving the modified Garcia scoring in infarcted rats (Fig. 5). tMCAO significantly decreased the maximum scoring from 18 points (sham-operated without tMCAO or hormones) to 7.5 points (tMCAO) in both sexes (Fig. 5A). The application of steroids preserved the scoring rates to 10.5 points (E), 12.5 points (P), and 11.9 points (E/P) in male rats. Similar findings with similar recovery rates were made in females (only E/P data are given). Exemplarily, the individual scoring performance for the six different tests is shown for males in Fig. 5B. Spontaneous activity, walking, and forepaw outstretching displayed best recovery rates, whereas sensory tasks showed only moderate or no reconstitution.

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Fig. 6. Effect of hormone treatment (25 lg E/10 mg P) on the number and morphology of Iba1-positive microglia cells in the penumbra of male rats 24 h after tMCAO. Panel (A) shows the quantitative evaluation of cell numbers and branching pattern on the ipsilateral side. Panel (B) shows representative sections demonstrating the morphological appearance of Iba1-positive microglial cells in the cerebral cortex penumbra on the contra- and ipsilateral side after tMCAO and E/P substitution. Panel (C) shows the expression and regulation of the activated microglia marker CD68 (ED 1) mRNA in the penumbra. ⁄⁄P 6 0.001 MCAO I vs. tMCAO c; #P 6 0.01 tMCAO i vs. E/P i. Data represent means ± SEM with n = 3 for morphological analysis/cell quantification and n = 5–6 for rtPCR gene expression study. E, 17b-estradiol; P, progesterone; n, number of tested individuals per group.

Using well-defined tissue landmarks representing the penumbra, we have assessed the attraction and morphology of microglia. Under control conditions and on the contralateral cerebral cortex, only few Iba1-positive microglial cells/macrophages with a small cell body and thin processes (o.e. ramified morphology) were detectable. In contrast, tMCAO significantly increased the number of Iba1-positive cells (120 cells/mm2, Fig. 6A). Cell bodies appeared swollen, processes were enlarged and retracted. After treatment with E/P, microglia accumulation within the penumbra was reduced to 80 cells/mm2. Signs of acute microglia activation, such as swollen somata and thickened processes were less evident in the hormone-treated group (Fig. 6B). Gene expression analysis for CD68, a specific marker for activated microglia, revealed almost no expression on the contralateral site of MCAO animals, whereas a 1000% increase was seen after MCAO on the ipsilateral site (Fig. 6C). Treatment with E/P attenuated this increase resulting in highly significant lower ipsilateral mRNA levels compared to MCAO alone. Gene and protein expression studies using tissues from the penumbra region of placebo and hormone-treated male rats 23 h after tMCAO were performed to obtain data about putative target genes/ proteins of E and P. Our analysis demonstrated that the tMCAOdependent increase of the T-cell marker CD3 and the induction of IL6 expression were significantly abolished by E/P on the mRNA level (Fig. 7A and B). Semi-quantitative Western blotting confirmed

these effects (Fig. 9A and B). CD3 protein was only detectable on the ipsilateral site after tMCAO and not present on the contralateral tMCAO site and after E/P exposure. Similarly, IL6 was de novo induced on the ipsilateral tMCAO site. E/P treatment strongly attenuated this effect. Expression of VEGF-A was up-regulated by tMCAO and further stimulated by E/P, whereas tMCAO-induced NGF expression was not affected by hormones (Fig. 7C and D). All three studied chemokines, CCL2, CCL3, and CCL5 were up-regulated by tMCAO. E/P inhibited the induction of CCL2 and CCL5 but further potentiated CCL3 expression (Fig. 8A–C). In addition, tMCAO enhanced the expression of ERa and -b but not PR within the cortical penumbra (Fig. 8D–F). This effect was neither abolished nor reinforced by hormone treatment. ELISA protein analysis revealed similar effects of hormone treatment on the amount of selected proteins as shown in the gene expression study (Fig. 9C). Hormone application significantly increased VEGF protein and reduced CCL2 and CCL5 proteins compared to tMCAO.

4. 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

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Fig. 7. Gene expression studies in tissue obtained from the male cerebral cortex penumbra after tMCAO and steroid substitution. (A) CD3 expression; (B) IL6 expression; (C) VEGF-A expression; (D) NGF expression. c, contralateral; CD, cluster of designation; E, 17b-estradiol; I, ipsilateral; IL, interleukin; n, number of tested individuals per group; NGF, nerve growth factor; P, progesterone; VEGF, vascular endothelial growth factor. ⁄P 6 0.01 tMCAO i vs. tMCAO c; ⁄⁄P 6 0.001 MCAO I vs. tMCAO c; #P 6 0.01 tMCAO i vs. E/P i. Data represent means ± SEM.

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 recently published studies (Acs et al., 2009; Lorenz et al., 2009). Nonetheless, P seemed to be slightly more effective when given alone compared to E in its protective capacity. Second, both sexes responded comparably to hormonal protection. Third, narrowing down of the penumbra region and exact tissue extraction allows to understanding the molecular and cellular processes occurring within the ‘‘tissue at risk’’. This modus operandi showed that the expression of genes controlling local inflammatory processes such as chemokines (CCL2 and CCL5), and interleukins (IL6) which are typically induced/increased after tMCAO are attenuated or inhibited. Additionally, the presence of CD3, Iba1, and CD68 typical markers for microglia/macrophages and lymphocytes, respectively, is reduced after E/P substitution compared to tMCAO. These two phenomena might be interpreted as steroid-mediated dampening of local microglia/macrophage, and lymphocyte attraction/activation (Denes et al., 2010a; Kriz, 2006; Wang et al., 2007). The induction of VEGF expression by E and P as shown here and also in a previous study using lung tissue (Trotter et al., 2009) may be more effective in the long-term outcome through augmentation of vascularization and capillary formation within damaged areas in the brain as suggested earlier (Ardelt et al., 2005). Few studies have focused on the role of P or a co-treatment with E compared to an exclusive E application on ischemic processes of acute cerebral ischemia in animal models. Individual E or P administration during or after stroke have been proven to protect cortical areas to a certain extent (Elzer et al., 2010; Gibson and Murphy, 2004; Lebesgue et al., 2009; Rusa et al., 1999). The lack of gonadal steroids in reproductively senescent female rats is supposed to be responsible for female predisposition of stroke and the degree of damage. Younger female rats display better protection against

stroke, i.e. a smaller infarct injury, compared to age-matched males (Alkayed et al., 2000). Supplementation of postmenopausal female rodents with reproductive steroid hormones mitigates ischemic brain injury (Alkayed et al., 2000). Since lesion volume calculation solely is a limited way of studying the beneficial effectiveness in rodents, we used functional outcome in conjunction with histopathological analysis in terms of assessing protective benefit. By concentrating on the penumbra tissue depicted by TTC-staining and NeuN-staining, we were able to show that the more dorsal parts of the cerebral cortex representing the transition zone are protected, whereas the lateral cerebral cortical parts appear to be less protected. Precisely, the major core part of the infarct area can be mainly assigned to the lateral cortex. This histopathological perspective is strongly supported by the behavioral results of the study which reveal motor skills engraved in the dorsal cortex to be better preserved compared to sensory functions located more laterally (Paxinos, 2004). It is noteworthy that all three chosen hormone groups and both sexes displayed similar good behavioral recovery rates after stroke. In a previous report, P has been shown to improve motor ability in mice up to several days after the infarct without demonstrating a clear-cut reduction of the infarct volume (Gibson and Murphy, 2004). In addition, E application meliorates hippocampus-associated cognitive functions after ischemic stroke (Söderström, 2009) and functional recovery of chronic behavior in MCAO mice (Li et al., 2004). Our results suggest that a combined hormone treatment appears to be the best choice to protect the cerebral cortex but not the caudate–putamen from ischemic damage. Nevertheless, P alone was nearly as effective as E and P together in reducing the lesion volume. At this point, we want to stress that the applied hormone doses prima facie appear relatively high. We attempted to yield steroid plasma concentrations corresponding to those known seen in pregnant females. To achieve these levels at a reasonable short time delay by the neck depot approach, such high doses are required. This can be deduced from our own dose–response studies shown in Figs. 3 and 4. Also previous substitution studies in adult mice (Acs et al., 2009; Ivanova and Beyer, 2003), pig (Trotter et al., 2006), and preterm children (Trotter et al., 1999) reveal that such high doses are necessary to obtain sustained and beneficial protective effects in vivo. Concerning the protective role of both steroids, similar observations were described using a hormone application strategy well before the onset of MCAO and lasting for at least 7 days (Toung et al., 2004). Both steroid hormones are neuroprotective against ischemic brain damage via a multitude of mechanisms (Arnold and Beyer, 2009; Kajta and Beyer, 2003; Liu et al., 2010a,b). Besides the importance of non-classical steroid signaling and its potency to interact with different intracellular cell survival and death signal cascades (Arnold and Beyer, 2009; Dhandapani and Brann, 2007; Kipp and Beyer, 2009; Lebesgue et al., 2009; Misiak et al., 2010), there is convincing evidence that E protects from ischemic injury by acting via classical ERs (Elzer et al., 2010; Dubal et al., 2006; Merchenthaler et al., 2003). After focal ischemia, the expression of ERs and thereby responsiveness of damaged tissue for E is locally induced showing early ERa and late ERb modulation (Dubal et al., 2006). We observed both ER subtypes to be up-regulated in the penumbra. Such an ischemia-autonomous tissue response may be seen in conjunction with known aromatase induction after MCAO in local astrocytes which occurs mainly one day after stroke and persists for several days (Carswell et al., 2005). The importance of astroglia for steroidmediated neuroprotection in the stroke area is well-accepted (Arevalo et al., 2009; Dhandapani and Brann, 2007). Interestingly, no regulation of PR was seen in our study or has been described so far in the literature. Irrespective of the steroid-induced signal transduction pathways, a key event in prevention of ischemia-

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Fig. 8. Gene expression studies in tissues obtained from the male cerebral cortex penumbra after tMCAO and steroid substitution. (A) CCL2 expression; (B) CCL5 expression; (C) CCL3 expression; (D) ERa expression; (E) ERb expression; (F) PR expression. c, contralateral; CCL, chemokine ligand; E, 17b-estradiol; ER, estrogen receptor; i, ipsilateral; n, number of tested individuals per group; P, progesterone; PR, progesterone receptor; ⁄P 6 0.01 tMCAO i vs. tMCAO c; ⁄⁄P 6 0.001 tMCAO i vs. tMCAO c; #P 6 0.01 tMCAO i vs. E/ P i. Data represent means ± SEM.

dependent neuronal degeneration is the control of local brainintrinsic inflammation mediated via immigrated peripheral lymphatic cells after blood–brain-barrier breakdown. P suppresses specific aspects of inflammatory responses such as attenuating TNFa, IL1b and TGFb induction after MCAO in mice likely due to nitric oxidase 3 regulation (Gibson et al., 2005), antagonizing NMDA receptor Ca2+ influx and activating Src–ERK signaling depending on the timing of hormone application before MCAO (Cai et al., 2008), increasing anti-apoptotic and decreasing proapoptotic molecules (Yao et al., 2005), preventing lipid oxidation (Roof et al., 1997), and suppressing ischemia-stimulated proliferation but improving survival of newborn neurons (Zhang et al., 2010a,b). In addition to the repeatedly described neuroprotective effects involving the regulation of inflammatory responses and reduction of Ca2+-dependent excitotoxic mechanisms which are extremely relevant for post-MCAO cellular reactions (reviewed by Lebesgue et al., 2009; Liu et al., 2009; Suzuki et al., 2009), more recent data show that E preserves endothelial function and mitochondrial integrity (Guo et al., 2010). This effect might account for the stabilization of the blood–brain-barrier (Liu et al., 2010a,b), inhibition of NADPH oxidase activation (Zhang et al., 2009), and regulation of thioredoxin activity in the brain thereby protecting from an excess of free radicals (Chen et al., 2010). Our study adds a novel aspect to the understanding of E- and P-dependent neuroprotection under ischemic condition. E and P selectively diminished MCAO-induced CCL2, CCL5, and IL6 expression and potentiated MCAO-dependent CCL3 mRNA levels in the penumbra. This is accompanied by a reduction of local Iba1-positive microglia invasion, proliferation and/or differentiation and by an abolishment of MCAO-mediated CD3 expression which represents a relevant marker for peripheral lymphocytes under acute ischemic conditions in the CNS (Perterfalvi et al., 2009). This assumption is further substantiated by the massive up-regulation of

CD63 expression after MCAO and attenuation of induction by E/ P in the penumbra. However, specific characterization of activated microglia is very difficult, as these cells share several antigens with different cell types including macrophages (Guillemin and Brew, 2004). In particular CD63 occurs in microglia and monocytes/macrophages. In addition to the overall statement that E/P diminishes local microglia/macrophage activation (demonstrated by dampening CD63 expression and reduction of Iba1 total cell numbers), we observed that E/P causes the shift from a swollen activated microglia to a more ameboid appearance which is almost indistinguishable from activated invading macrophages (Guillemin and Brew, 2004; Tambuyzer et al., 2009). This suggests the possibility that sex hormones might promote selectively the activation of microglia but coevally inhibit the mobilization of macrophages. To date, our data cannot answer this question. Further studies are required. Besides cytokines, local ‘‘early’’ chemokines and prostanoids secreted either by astroglia and/or damaged neurons are major biological factors which direct leukocytes to sites of brain injury and determine microglia attraction and their morphological reorganization associated with inflammatory activity (Babcock et al., 2003; Cross and Woodroofe, 1999; Gao and Ji, 2010; Johann et al., 2008; Ziebell and Morganti-Kossmann, 2010). These chemokines are also detectable in the peri-lesioned region of cerebral tissue of patients with post-traumatic brain contusion (Stefini et al., 2008) and contribute in co-operation with cytokines to the size of infarct lesion (Denes et al., 2010a). A particular role in the regulation of local inflammatory networks after brain damage and acute focal ischemia is assigned to the chemo-attractant protein CCL2 (Conductier et al., 2010; Semple et al., 2010). It has been recently established that CCL2 (monocyte chemoattractant protein 1) is necessary for recruiting blood-borne cells to the injury site (Schilling et al., 2009). Furthermore, CCL2-deficient (/) mice showed a reduced secondary post-traumatic

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Fig. 9. (A and B) Semi-quantitative Western blot analysis of tissues from the male cerebral cortex penumbra after tMCAO and steroid substitution for CD3 and IL6. Insets in (A) and (B) show representative blots. No statistical analysis and quantification could be performed due to zero (n.d.) values on the reference site or E/P values. (C) ELISA of tissues from the male cerebral cortex penumbra after tMCAO and steroid substitution for CCL2, CCL5, VEGF and IL6. c, contralateral; CCL, chemokine ligand; CD, cluster of designation; E, 17b-estradiol; I, ipsilateral; IL, interleukin; n, number of tested individuals per group; n.d., not detectable; P, progesterone; VEGF, vascular endothelial growth factor; ⁄P 6 0.01 tMCAO i vs. tMCAO c; #P 6 0.01 tMCAO i vs. E/P I; ##P 6 0.001 tMCAO i vs. E/P i. Data represent means ± SD.

brain damage illustrated by a reduced lesion volume, macrophage accumulation, and astrogliosis (Semple et al., 2010). Less valuable information about the role of CCL5 (RANTES) during stroke is available. CCL5 expression precedes the appearance of immune cells and appears to be involved in the early regulation of immune responses in immature rats (Bona et al., 1999). Chronic infections exacerbate ischemic neural damage and potentiate cerebral microvascular dysfunction through CCL5-dependent systemic inflammatory responses (Denes et al., 2010b; Terao et al., 2008). Besides its well-established role for atherosclerosis and as a genetic risk factor, data detailing the direct function of IL6 during an acute stroke scenario and IL6 regulation in the brain by gonadal steroids are flimsy (Yamada et al., 2008; Vikman et al., 2007). Only recently, E was found to inhibit the rise in CCL5 brain levels after the systemic application of lipopolysaccharides to provoke systemic inflammation (Brown et al., 2010). There exists an abundance of information concerning the neuroprotective potential of both sex hormones under ischemic conditions in the CNS. Yet, the present study adds several unique and novel aspects to this topic. An important observation is the obvious cell protection and tissue preservation irrespective of the gender and the coherent impeding of behavioral dysfunction. Although this observation refers to short-term outcome after 24 h, preliminary data from our group now show that recovery/ protection from ischemia by steroids is also verifiable and at a similar range after one week. Again, this is only seen if both steroids are applied together but not conclusively in single applica-

tions. Thus, we assume that steroid application soon after the onset of ischemia at high concentrations stabilizes neuronal activity/performance until reperfusion is sufficient to supply neurons again with vital agents. Although our study thitherto provides only correlative data, we assume that the interaction and fine tuning of the local inflammatory scenario is essential for the implementation of long-term protection. This includes dampening local microglia responses and lymphocyte attenuation most likely through the control of astroglial chemokine/interleukin production. CCL2 and CCL5 appear to be major players in this interplay and targets of steroid action. We are presently studying CCL2 and CCL5 knockout animals in the context of neuroprotection after tMCAO, and we have recently created a double knockout mouse for both chemokines. Future studies will show whether this chemokine strategy will help us to better understand steroid-related neuroprotective mechanisms.

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

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