ABSTRACT
Title
Simvastatin modulates autophagy in neonatal hypoxia-ischemia
Authors
S. Girelli1, S. Carloni1, G. Buonocore2, M. Longini2 and W. Balduini1
1Dept. of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy
2Dept. of Paediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy
1Dept. of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy
2Dept. of Paediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy
Abstract
We have previously shown that the prophylactic treatment with simvastatin (Sim) has long-lasting neuroprotective effects in neonatal hypoxia-ischemia (HI). Sim improved the brain damage and the long-lasting neurological outcomes induced by HI (Balduini et al., 2001), and neuroprotection was associated with reduction of inflammatory cytokines expression, caspase-3 activation and apoptotic cell death (Carloni et al., 2006). These effects were concomitant with the activation of the survival pathways PI3K/Aktand CREB (Carloni et al., 2009). Recently, we reportedthat the activation of these survival pathways is strictly connected to the activation of the autophagy process (Carloni et al., 2010). The present study was undertaken to evaluate if autophagy was also involved in the neuroprotective effect of Sim. Furthermore, since autophagy, apoptosis, and necrosis are interrelated phenomena, we also evaluated if the statin could reduce HI-induced necrotic cell death.
On postnatal-day 7, rats were subjected to permanentligation of the right common carotid artery followed by 2.5 h hypoxia. Sim (20 mg/kg) was administered from postnatal day 1 to day 7 before the onset of the ischemic procedure. Animals were sacrificed 2 or 24 hours after HI for biochemical studies.To assess the activation of autophagy and necrotic cell death, animals were treated with monodancylcadaverine (MDC, 1,5 mg/kg i.p. for in vivo labeling of autophagic vacuoles) or propidium iodide (PI, half microliter, 1 mg/mL icv), respectively.
Beclin-1 expression, a marker of autophagy, was increased in the lesioned side of ischemic animals and treatment with Sim further increased its expression. MDC labelling was observed in the lesioned side of both vehicle treated and Sim-treated ischemic animals. MDC labeling co-localized with LC3 II, a microtubule-associated protein that is lipidated upon activation of autophagy. MDC labeling was observed 2 hours after HI and was higher at 24 hours. Treatment with Sim significantly increased the MDC labeling at both the time points evaluated. Sim also reduced necrotic cell death as revealed by the counting of PI-positive cells and the hematoxylin-eosin staining.
These results indicate that prophylactic treatment with Sim significantly increases autophagy and reduces necrotic cell deathin this model of neonatal HI. We suggest that in this model the statin may cause pharmacological preconditioning of which the increased autophagy flux may be an important component.
Balduini et al. (2001). Stroke. 32 :2185-91.
Carloni et al. (2006). Neurobiol Dis. 21 :119-26.
Carloni et al. (2009). Exp Neurol. 220 :82-9.
Carloni et al. (2010). Autophagy. 6 :366-77.
On postnatal-day 7, rats were subjected to permanentligation of the right common carotid artery followed by 2.5 h hypoxia. Sim (20 mg/kg) was administered from postnatal day 1 to day 7 before the onset of the ischemic procedure. Animals were sacrificed 2 or 24 hours after HI for biochemical studies.To assess the activation of autophagy and necrotic cell death, animals were treated with monodancylcadaverine (MDC, 1,5 mg/kg i.p. for in vivo labeling of autophagic vacuoles) or propidium iodide (PI, half microliter, 1 mg/mL icv), respectively.
Beclin-1 expression, a marker of autophagy, was increased in the lesioned side of ischemic animals and treatment with Sim further increased its expression. MDC labelling was observed in the lesioned side of both vehicle treated and Sim-treated ischemic animals. MDC labeling co-localized with LC3 II, a microtubule-associated protein that is lipidated upon activation of autophagy. MDC labeling was observed 2 hours after HI and was higher at 24 hours. Treatment with Sim significantly increased the MDC labeling at both the time points evaluated. Sim also reduced necrotic cell death as revealed by the counting of PI-positive cells and the hematoxylin-eosin staining.
These results indicate that prophylactic treatment with Sim significantly increases autophagy and reduces necrotic cell deathin this model of neonatal HI. We suggest that in this model the statin may cause pharmacological preconditioning of which the increased autophagy flux may be an important component.
Balduini et al. (2001). Stroke. 32 :2185-91.
Carloni et al. (2006). Neurobiol Dis. 21 :119-26.
Carloni et al. (2009). Exp Neurol. 220 :82-9.
Carloni et al. (2010). Autophagy. 6 :366-77.