NCX1 Expression and Functional Activity Increase in Microglia Invading the Infarct Core
Background and Purpose— The sodium–calcium exchanger NCX1 represents a key mediator for maintaining [Na+]i and [Ca2+]i in anoxic conditions. To date, no information is available on NCX1 protein expression and activity in microglial cells under ischemic conditions.
Methods— By means of Western blotting, patch-clamp electrophysiology, single-cell Fura-2 acetoxymethyl-ester microfluorometry, immunohistochemistry, and confocal microscopy, we investigated the regional and temporal changes of NCX1 protein in microglial cells of the peri-infarct and core regions after permanent middle cerebral artery occlusion. The exchanger expression and activity were measured in primary microglia isolated ex vivo from the core region of adult rat brains 7 days after permanent middle cerebral artery occlusion and in cultured microglia under in vitro hypoxia.
Results— One day after permanent middle cerebral artery occlusion, NCX1 protein expression was detected in some microglial cells adjacent to the soma of neurons in the infarct core. More interestingly, 3 and 7 days after permanent middle cerebral artery occlusion, NCX1 signal strongly increased in the round-shaped microglia invading the infarct core. Cultured microglial cells obtained from the core also displayed increased NCX1 expression as compared with contralateral cells and showed enhanced NCX activity in the reverse mode of operation. Similarly, NCX activity and NCX1 protein expression were significantly enhanced in BV2 microglia exposed to oxygen and glucose deprivation, whereas NCX2 and NCX3 were downregulated. Interestingly, in NCX1-silenced cells, [Ca2+]i increase induced by hypoxia was completely prevented.
Conclusion– The upregulation of NCX1 expression and activity observed in microglia after permanent middle cerebral artery occlusion suggests a relevant role of NCX1 in modulating microglia functions in the postischemic brain.
Microglial cells are one of the first nonneuronal cells that respond to ischemic injury and in which, like occurs in most immune cells, the initiation of cellular responses, including morphological changes, proliferation, motility, phagocytosis, and increased synthesis of receptors and secretory products, involves modifications of calcium homeostasis.1,2⇓ In particular, long-lasting ionic alterations occurring in microglia in response to the ischemic injury play a crucial role in inducing and maintaining neurotrophic, inflammatory, and remodeling responses in the postischemic brain.3
The sodium–calcium exchanger (NCX), an integral protein belonging to the superfamily of plasma membrane exchangers, represents a key mediator for maintaining intracellular [Na+]i and [Ca2+]i homeostasis in response to brain ischemia. Indeed, depending on the levels of [Na+]i and [Ca2+]i, NCX can operate either in the forward mode, coupling the uphill extrusion of Ca2+ to the influx of Na+ ions, or in the reverse mode, coupling the extrusion of Na+ to the influx of Ca2+ ions.4,5⇓
Three NCX genes, that is, NCX1, NCX2, and NCX3, have been identified and cloned.6–8⇓⇓ NCX1 is broadly expressed in the heart, brain, and kidney, whereas NCX2 and NCX3 are exclusively expressed in the brain and skeletal muscle.7,8⇓
Among these 3 genes, the ubiquitous NCX1 is the most highly characterized member.9 By contrast, in the brain, although isoform-specific cellular expression patterns suggest distinct functions for each of the 3 exchangers,10 the physiological roles of the NCX1 gene product have not yet been as well defined as those in the heart.
In vitro studies provided evidence that, among the different NCX genes, NCX1 is the most highly expressed in microglia11,12⇓; interestingly, the direct exposure of cultured microglia to interferon-γ or nitric oxide13,14⇓ enhanced NCX1 transcripts levels. However, no information is yet available on NCX1 protein expression and activity in microglia under ischemic conditions. Recently, by means of radioactive in situ hybridization experiments, our research group showed that NCX1 transcripts display a different expression pattern in the ischemic core and in the peri-infarct regions after permanent middle cerebral artery occlusion (pMCAO) in rats15; whether these modifications also occur in microglia in the postischemic brain still remains unknown.
In the present study, by means of immunohistochemistry, double immunofluorescence, and confocal microscopy, we first investigated the regional and temporal changes of NCX1 protein in microglia of the core and peri-ischemic areas 1, 3, and 7 days after pMCAO as compared with the contralateral undamaged area. Then, by means of Western blotting, electrophysiology, and single-cell Fura-2 acetoxymethyl-ester microfluorometry, we explored NCX1 expression and NCX activity ex vivo, in primary glial cultures dissociated from ischemic adult rat brain 7 days after pMCAO and in vitro, in microglia exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation.
Materials and Methods
Male Sprague-Dawley rats (250 to 270 g, n=45; Charles River; Calco, Italy) were housed in a temperature- and humidity-controlled colony room under diurnal/lighting conditions. Animal handling was in accordance with the International Guidelines for Animal Research and the experimental protocol was approved by the Animal Care and Use Committee of “Federico II” University of Naples.
Rats were divided into 3 groups: (1) control (n=5); (2) sham-operated (n=5); and (3) ischemic (n=5). The latter received pMCAO for 1, 3, and 7 days, respectively. pMCAO was performed as previously described.15,16⇓ A 2-cm incision was made between the orbit and the ear and another to divide the temporal muscle. Next, a small window (diameter=2 mm) was made just over the visibly identified middle cerebral artery. The left pMCAO was performed by electrocoagulation with a bipolar electrocauterizer (Diatermo). The pMCAO insult was performed as close as possible to the middle cerebral artery origin, that is, near the circle of Willis. The body temperature was monitored with a rectal probe and maintained at 37±0.5°C until awakening. Sham-operated animals underwent the same procedures except for middle cerebral artery electrocoagulation. In some of the experimental and sham-operated rats, a catheter was inserted into the femoral artery to measure arterial blood gases with a blood gas analyser (Chiron Diagnostic). Cerebral blood flow was monitored in the cerebral cortex ipsilateral to the occluded middle cerebral artery with a laser Doppler flowmeter.16,17⇓
Glial Cell Cultures Obtained From the Ischemic Brain
Seven days after pMCAO, the tissue harvested from the cortical core and the corresponding contralateral cortex was first dissociated enzymatically in a solution containing 0.125% trypsin and 1.5 mg/mL DNase (Sigma) and then mechanically in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, 2 mmol/L l-glutamine (Gibco). Cell pellet was resuspended and plated onto poly-d-lysine-coated coverslips. Because we did not observe significant changes in terms of cell morphology or NCX activity at 1 day in vitro if compared with 3 days in vitro, all experiments were performed at 3 days in vitro. Only after 7 days in vitro, the IB4-positive cells obtained from the core gradually returned to a more ramified morphology (data not shown).
BV2 Microglia and OGD
The BV2 microglial cell line was generously provided by Prof G. M. Lauro (University of Rome “Roma Tre”). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, and 2 mmol/L l-glutamine. Hypoxia was simulated by exposing cells to the culturing medium devoid of glucose and serum and previously saturated with 95%N2/5% CO2, as described.18 Then, they were placed in an air-tight box chamber (Billups-Rothenberg Inc) in which the atmosphere was also saturated with 95% N2/5% CO2. After 40 minutes of incubation at 37°C, cells were transferred to culturing medium and returned to the incubator under normoxia. Cells were reoxygenated for 6, 24, and 48 hours. Control cells were kept under normoxia.
Nitric oxide (NO) generation in BV2 cells was determined using the Griess reaction assay.19
Protein samples were separated on 8% or 14% polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes. Filters were incubated with mouse monoclonal anti-NCX1 (1:500; Swant), rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba1; 1:2000; Wako), rabbit polyclonal anti-NCX2 (1:200; Primm, Italy), rabbit polyclonal anti-NCX3 (1:3000; kindly provided by Dr K. D. Philipson and Dr D. A. Nicoll, Los Angeles, Calif), and mouse monoclonal anti-β-actin (1:1000; Sigma). Immunoreactive bands were detected using the chemiluminescence system (Amersham-Pharmacia-Biosciences).
Immunostaining and confocal immunofluorescence procedures were performed as previously described.10,15,20⇓⇓ The animals were euthanized 1, 3, and 7 days after pMCAO onset. The rats were anesthetized intraperitoneally with chloral hydrate (300 mg/kg) and perfused transcardially with 4% wt/vol paraformaldehyde and 15% wt/vol picric acid in phosphate buffer. The brains were sectioned coronally at 60 μm on a vibratome. After blocking, sections were incubated with the following primary antisera: anti-NCX1 (1:500), anti-NeuN (mouse monoclonal, 1:2000; Chemicon), and anti-Iba1 (1:2000). Microglia were also identified using the isolectin-B4 conjugated to fluorescein isothiocyanate (isolectin-B4 conjugated to fluorescein isothiocyanate; 1:200). After incubations with biotinylated secondary antibodies, the peroxidase reaction was developed using 3,3′-diaminobenzidine/4-HCl as a chromogen. For double immunofluorescence, sections were instead incubated in a mixture of the fluorescent-labeled secondary antibodies (Alexa 488/Alexa 594-conjugated antimouse/antirabbit IgG). Because no immunoreactivity differences were found between control and sham-operated animals, the data are presented as a comparison of pMCAO-bearing and sham-operated rats. Cell cultures were fixed in 4% wt/vol paraformaldehyde in phosphate buffer for 30 minutes and incubated with primary antisera as described. Subsequently, they were incubated in a mixture of Alexa 594-conjugated antimouse-IgG and isolectin-B4 conjugated to fluorescein isothiocyanate (1:200). Images were observed using a Zeiss LSM510 META/laser scanning confocal microscope. Single images were taken with an optical thickness of 0.7 μm and a resolution of 1024×1024.
[Ca2+]i was measured by single-cell Fura-2 acetoxymethyl-ester videoimaging, as previously described.19 Primary glial cultures were first preincubated with isolectin-B4 conjugated to fluorescein isothiocyanate for 4 to 6 hours to identify microglia, and, at the end of this incubation, the cells were loaded with 6 μmol/L Fura-2 acetoxymethyl-ester for 30 minutes at 37°C in normal Krebs solution.19 Then, the coverslips were placed into a perfusion chamber (Medical System, Co, Greenvale, NY) mounted onto the stage of an inverted Zeiss Axiovert 200 microscope (Carl Zeiss) equipped with a FLUAR 40× oil objective lens. [Ca2+]i was simultaneously measured together with IB4 fluorescence in cells alternatively illuminated at 340 nm/380 nm and 490 nm wavelengths. NCX activity was evaluated as Ca2+ uptake through the reverse mode by switching the normal Krebs medium to Na+-deficient NMDG+ medium (Na+-free).
INCX was recorded from BV2 microglia and primary glial cell cultures by the patch-clamp technique in whole-cell configuration as previously described.21 Ringer solution contained 20 mmol/L tetraethylammonium, 1 mmol/L 4-aminopiridine, 50 nmol/L tetrodotoxin, 10 μmol/L nimodipine, and 50 μmol/L Ba2+ to block tetraethylammonium- and 4-aminopiridine-sensitive K+, tetrodotoxin-sensitive Na+, l-type Ca2+, and Kir currents.
Silencing of NCX1 in BV2 cells was performed with HyPerfect-Transfection Kit (Qiagen) by using 2 different FlexiTube siRNA for NCX1: (1A) Mm_Slc8a1_1 (5′-AAGGATTCTGAAGGAACTTAA-3′); and (1B) Mm_Slc8a1_4 (5′-CTGGCTCATATTACTGTAAGA-3′) and the validated irrelevant Allstars short-interfering RNA as a negative control (Qiagen). Cells were incubated with OptiMEM (Invitrogen) supplemented with RNAiFect Transfection Reagent (Qiagen) and 20 nM of each short-interfering RNA duplex for 15 hours. Then, cells were incubated in culturing medium for an additional 72 hours. Protein extracts were subjected to Western blot analysis with the anti- NCX1 antibodies.
Data are expressed as mean±SEM of values obtained in 3 separate experiments. Statistical comparisons between controls and treated groups were performed using the one-way analysis of variance followed by Newman Keul’s test. P<0.05 was considered significant.
NCX1 Distribution 1 Day After pMCAO
At Day 1, a marked loss of NeuN immunoreactivity was evident in the deep cortical layers (Figures 1A, a–h and 1B, a, e). The pattern of immunoreactivity observed with the anti-NCX1 antibody was identical in the unaffected hemisphere and in sham-operated animals (Figure 1A, i–p), in agreement with previous studies performed with the same antibody.10,22⇓ In the somatosensory cortex of ischemic animals, NCX1 immunoreactivity of the neuropil was less intense than in the unaffected hemisphere (Figure 1B, b–f). Nonetheless, it was clearly detectable in coiled apical dendrites, some of which appeared intensely immunostained (Figures 1B, c–g and 1G). In addition, the NCX1 signal became evident within the perikaryon of neurons located in the deep cortical layers of the infarct zone (Figure 1B, d–h). Interestingly, in the same region, NCX1 immunoreactivity was also confined around the soma of neuronal profiles, possibly recognizing perineuronal glial cells (Figure 1C, a–c). Confocal immunofluorescence experiments revealed that in the infarct core, some perineuronal microglial cells were double-labeled for the microglial Iba1 marker and NCX1 (Figure 1C, d–f). Intriguingly, NCX1 immunoreactivity colocalized with Iba1 in cells located near the vessel wall, thus suggesting NCX1 expression in perivascular macrophages (Figure 1C, g–i). By contrast, in the contralateral hemisphere or in sham-operated animals, the anti-NCX1 antibody did not stain NCX1 immunoreactivity in microglia (not shown).
NCX1 Distribution 3 Days After pMCAO
Three days after pMCAO, when the loss of NeuN staining in the deep cortical layers of the infarct region was more pronounced (Figures 2A, a–h and 2B, a, e), NCX1 immunosignal increased in this region, particularly within the medial and superficial region of the somatosensory cortex, where its expression was strongly evident in beaded apical dendrites (Figure 2B, b, d). More interestingly, many round-shaped cells invading the infarct area and resembling macrophage-like morphology exhibited intense NCX1 immunoreactivity (Figure 2B, f–h), whereas in the corresponding contralateral area, no NCX1 immunosignal was evident (not shown). Double-labeled experiments revealed that NCX1 was selectively coexpressed in the round Iba1-positive microglia invading the infarct core (Figure 2C, a–f). In the peri-ischemic region, outside the infarct area, dendrites positive for NCX1 appeared intensely immunostained and several cells showed clear NCX1 immunoreactivity (Figure 2B, b–c). No colocalization between NCX1 immunoreactivity and other phenotypes of Iba1-positive microglia were detected in the peri-ischemic region (not shown).
NCX1 Distribution 7 Days After pMCAO
At Day 7, when the core was largely devoid of NeuN signal (Figures 3A, a–h and 3B, a, d), NCX1 immunoreactivity in the neuropil was strongly decreased (Figure 3A, i–p). By contrast, the NCX1 immunosignal largely increased in round-shaped cells located in the core (Figure 3B, e–f). Confocal immunofluorescence experiments revealed that Iba1-positive cells accumulating in the infarct core at 7 days were much more abundant than those observed in the same area at 3 days. Interestingly, only those Iba1-positive microglial cells that showed round-shaped morphology coexpressed NCX1 immunosignal (Figure 3C, a–i). In the more central region of the infarct core, NCX1/Iba1 double-labeled cells represent approximately 80% of Iba1-positive cells (Figure 3C, g–i). In the peri-ischemic region, the exchanger immunosignal appeared still increased (Figure 3B, b–c), as already observed 3 days after pMCAO, but no colocalization between NCX1 and Iba1 immunoreactivities was found (not shown).
Upregulation of NCX1 Expression in Microglia Obtained From the Core Region
Double-labeling of NCX1 protein with the microglial marker IB4 revealed that NCX1 immunoreactivity in microglia isolated from the contralateral region was barely detectable (Figure 4A–F). The majority of cells (91.5±1.5) isolated from the contralateral cortex were IB4(+), and only 12.1%±4.2% of them express NCX1 protein (Figure 4M). By contrast, microglia isolated from the core strongly expressed NCX1 immunoreactivity (Figure 4G–L). Quantitative analysis revealed that 46%±3.6% of cells isolated from the core were IB4(+). Among them, 94.2%±4.8% were also positive for NCX1. In these cells, NCX1 signal was intensely expressed on the plasma membrane (Figure 4G–L).
Upregulation of NCX Activity in Microglia Obtained From the Core Region
To assess whether a modulation of NCX function peculiarly occurred in microglial cells of the ischemic core, its activity was assessed by using Na+-dependent [Ca2+]i increase monitored by FURA-2 microfluorometry and patch clamp in a whole-cell configuration either in IB4(+) cells or IB4(−) cells (Figure 5). In addition, NCX activity was assessed in IB4(+) cells from the corresponding contralateral cortex (Figure 5E, G). The perfusion of Na+-free solution was followed by a fast linear rise in [Ca2+]i that was largely greater in IB4(+) cells obtained from the core if compared with both IB4(−) cells of the same ischemic region and IB4(+) cells obtained from the corresponding contralateral area (Figure 5E–G). Accordingly, INCX, recorded in the reverse and forward mode of operation, were significantly higher in IB4(+) cells obtained from the core if compared to those recorded both in IB4(−) cells of the same region and IB4(+) cells of the contralateral hemisphere. Relevantly, in microglia obtained from the core, the current carried by NCX working in the reverse mode was much higher than that recorded in the forward mode (Figure 5F–G).
Upregulation of NCX1 Protein Expression and NCX Activity in BV2 Microglia Exposed to OGD
To dissect the direct effects of hypoxic conditions on NCX1 expression/activity when microglial cells are incubated alone, we used a more simple system constituted by BV2 microglial cell line exposed to OGD followed by reoxygenation.
When BV2 microglia were exposed to OGD, a significant increase in NCX1 protein expression occurred 24 hours after reoxygenation; after 48 hours of reoxygenation, NCX1 protein levels were still higher than those in normoxic cells, although they were reduced if compared with those measured at 24 hours. Conversely, after 6 hours, no variations in NCX1 protein expression were detected (Figure 6A). When BV2 microglia were exposed to OGD, at variance with NCX1 expression, a significant decrease in both NCX2 and NCX3 occurred both at earlier and later times of reoxygenation (Figure 6B–C). Furthermore, OGD/reoxygenation also caused an increase in Iba1 protein expression and NO production in microglia (Figure 6D). The increase in these 2 hallmarks indicates that OGD activates microglia in vitro.23
In microglia exposed to OGD plus 6 and 24 hours of reoxygenation, isolated INCX, recorded both in the forward and reverse modes of operation, were significantly higher than those detected under normoxia. After 48 hours of reoxygenation, INCX declined but was nonetheless still higher than that recorded in normoxic cells (Figure 6E–F). Accordingly, [Ca2+]i, significantly increased 6 hours after reoxygenation if compared with normoxic levels. After 24 hours of reoxygenation, although [Ca2+]i decreased, it was higher than that of control. Interestingly, after 48 hours of reoxygenation, [Ca2+]i returned to basal normoxic values (Figure 6G).
NCX1 Silencing Prevents OGD-Induced [Ca2+]i Increase in BV2 Microglia
To examine whether the altered Ca2+ handling was specifically related to NCX1 expression/activity increase, we knocked down NCX1 using 2 selective short-interfering RNA (1A and 1B) in BV2 microglia. Both short-interfering RNAs significantly reduced NCX1 protein in microglia, as revealed by Western blot (Figure 6H, upper panel). Consistently, in NCX1-silenced cells, NCX activity, measured as Na+-free induced [Ca2+]i increase (ie, reverse mode), was significantly reduced (Figure 6H, lower panel). Interestingly, when NCX1-silenced BV2 cells were exposed to OGD plus 6 hours of reoxygenation, the increase in [Ca2+]i, was completely prevented (Figure 6I). This finding, together with the results showing that the protein expression of the other 2 NCX isoforms—NCX2 and NCX3—significantly decreased after OGD, underlined the relevant role played by the NCX1 isoform in round phagocytic microglial cell during hypoxic conditions.
The present study, by means of in vivo, ex vivo, and in vitro integrative approaches, demonstrated that NCX1 expression and activity are upregulated in microglia in response to the ischemic injury. In particular, 1 day after pMCAO, NCX1 protein expression was detected only in some microglial cells located adjacent to the soma of neurons in the infarct core. More interestingly, 3 and 7 days after pMCAO, NCX1 signal progressively increased in the Iba1-positive microglia invading the infarct core. In these cells, NCX1 expression was limited to the round phagocytic phenotype, which represents the final stages of microglia activation.2,23⇓ Interestingly, microglial cells isolated from the lesioned core region 7 days after pMCAO and cultured in vitro intensely expressed NCX1 protein. Furthermore, NCX activity in the reverse mode of operation significantly and selectively increased in these cells. Accordingly, we found that the BV2 microglial cell line, when exposed to anoxic conditions, displayed a significant increase of INCX and of NCX1, but not of NCX2 and NCX3 protein expression that were in contrast downregulated. The enhanced NCX activity may explain the modifications of [Ca2+]i observed at different time intervals in microglial cells after OGD. In particular, the increase in [Ca2+]i detected at 6 hours after OGD might be attributed to an increased activity of NCX1 in the reverse mode of operation, because NCX1 silencing fully prevented the OGD-induced [Ca2+]i rise. Consistently with this interpretation, in other pathological conditions such as interferon-γ or NO exposure, the activity and expression of NCX increased in microglia in the reverse mode of operation, causing Na+-dependent Ca2+ uptake.14 The presence of NCX1 signal in microglial cells invading the infarct core suggests that this exchanger, by determining an increase of [Ca2+]i, might play an important role in calcium-mediating microglia functions.2 Recently, Ifuku et al24 showed that the Ca2+ influx through reverse-mode activity of the Na+/Ca2+exchanger is a prerequisite for bradykinin- and B1-agonist-induced microglial motility. In these cells, the knocking out of NCX1 in the heterozygous state impairs bradykinin-induced chemotaxis or migration in microglia.
Another aspect that deserves consideration is that the increase in NCX activity in the BV2 microglial cell line was already detected when NCX1 protein upregulation was not yet observed. This early increase of NCX activity can be due to the augmented levels of NO observed in the present study in anoxic microglia. In fact, it has been shown that NO exerts a stimulatory action on NCX operating in the reverse mode14 and the time course of the production of this gaseous mediator followed the same pattern as that observed for INCX, detected by the patch clamp technique. The intense expression of NCX1 in microglia suggests its functional involvement in regulating the activity of these cells in the postischemic brain. In fact, microglia serve as scavenger cells in the injured brain. Indeed, phagocytic clearance of structures that have lost their function such as necrotic debris of cells, dendrites, and myelin is considered beneficial and represents a prerequisite for repair attempts in cerebral ischemia.2 However, the marker Iba1 does not allow us to discriminate whether NCX1/Iba1 double-labeled cells observed 7 days after pMCAO belongs to round phagocytic microglia or circulating bloodborne brain macrophages invading the infarct core. Whether the increase in NCX1 expression and activity could contribute to the beneficial actions exerted by microglial activity during the different phases of microglia activation after ischemia cannot be determined from the present study. However, the importance of NCX1 function in the brain has been already highlighted by recent findings showing that under cerebral ischemia, the specific knocking down of the ncx1 gene as well as ncx2 and ncx3 dramatically increases the extent of the ischemic lesion in rats and mice.16,21,25⇓⇓ This suggests that the increased activity of NCX1 in microglia of the postischemic brain might exert a protective role.
Collectively, the upregulation of NCX1 expression and activity observed in microglial cells after pMCAO suggests the relevant role played by NCX1 in modulating microglial functions in the postischemic brain. A thorough understanding of NCX1 function in microglia may help to provide novel disease-modifying approaches in cerebral ischemia.
We thank Prof K. D. Philipson and Dr D. A. Nicoll (University of California, Los Angeles) for providing anti-NCX3 antibodies; Prof G. M. Lauro (University of Rome “Roma Tre”) for providing BV2 microglial cell line; Dr Paola Merolla for the editorial revision; and Antonella Di Crisci, Carla D’Avanzo, Alba Esposito, and Pellegrino Lippiello for technical assistance.
Sources of Funding
This work was supported by grants from COFIN 2006, “Ministero Affari Esteri, Direzione Generale per la Promozione e la Cooperazione Culturale Fondi Italia-Cina Legge 401/1990 2007, 2008,” Ministero della Salute, Ricerca Sanitaria RF-FSL352059 Ricerca finalizzata 2006; Ministero della Salute, Ricerca Oncologica 2006; Ministero della Salute, Progetto Strategico, 2007; and Ministero della Salute, Progetto Ordinario, 2007 (to L.A.).
- Received May 8, 2009.
- Accepted May 26, 2009.
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