Elga Esposito

Transfer of mitochondria from astrocytes to neurons after stroke

Neurons can release damaged mitochondria and transfer them to astrocytes for disposal and recycling. This ability to exchange mitochondria may represent a potential mode of cell-to-cell signalling in the central nervous system. Here we show that astrocytes in mice can also release functional mitochondria that enter neurons. Astrocytic release of extracellular mitochondrial particles was mediated by a calcium-dependent mechanism involving CD38 and cyclic ADP ribose signalling. Transient focal cerebral ischaemia in mice induced entry of astrocytic mitochondria into adjacent neurons, and this entry amplified cell survival signals. Suppression of CD38 signalling by short interfering RNA reduced extracellular mitochondria transfer and worsened neurological outcomes. These findings suggest a new mitochondrial mechanism of neuroglial crosstalk that may contribute to endogenous neuroprotective and neurorecovery mechanisms after stroke.

NCX1 Is a Novel Target Gene for Hypoxia-Inducible Factor-1 in Ischemic Brain Preconditioning

Background and Purpose— The sodium–calcium exchanger-1 (NCX1) represents a key mediator for maintaining [Na+]i and [Ca2+]i homeostasis. Although changes in NCX1 protein and transcript expression have been detected during stroke, its transcriptional regulation is still unknown. Thus far, however, there is evidence that hypoxia-inducible factor-1 (HIF-1) is a nuclear factor required for transcriptional activation of several genes implicated in stroke. The main objective of this study was to investigate whether NCX1 gene might be a novel target of HIF-1 in the brain. Methods and Results— Here we report that: (1) in neuronal cells, NCX1 increased expression after oxygen and glucose deprivation or cobalt-induced HIF-1 activation was prevented by silencing HIF-1; (2) the brain NCX1 promoter cloned upstream of the firefly-luciferase gene contained 2 regions of HIF-1 target genes called hypoxia-responsive elements that are sensitive to oxygen and glucose deprivation or cobalt chloride; (3) HIF-1 specifically bound hypoxia-responsive elements on brain NCX1, as demonstrated by band-shift and chromatin immunoprecipitation assays; (4) HIF-1&agr; silencing prevented NCX1 upregulation and neuroprotection induced by ischemic preconditioning; and (5) NCX1 silencing partially reverted the preconditioning-induced neuroprotection in rats. Conclusions— NCX1 gene is a novel HIF-1 target, and HIF-1 exerts its prosurvival role through NCX1 upregulation during brain preconditioning.

The NCX3 isoform of the Na+/Ca2+ exchanger contributes to neuroprotection elicited by ischemic postconditioning

It has been recently shown that a short sublethal brain ischemia subsequent to a prolonged harmful ischemic episode may confer ischemic neuroprotection, a phenomenon termed ischemic postconditioning. Na+/Ca2+ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, are plasma membrane ionic transporters widely distributed in the brain and involved in the control of Na+ and Ca2+ homeostasis and in the progression of stroke damage. The objective of this study was to evaluate the role of these three proteins in the postconditioning-induced neuroprotection. The NCX protein and mRNA expression was evaluated at different time points in the ischemic temporoparietal cortex of rats subjected to tMCAO alone or to tMCAO plus ischemic postconditioning. The results of this study showed that NCX3 protein and ncx3 mRNA were upregulated in those brain regions protected by postconditioning treatment. These changes in NCX3 expression were mediated by the phosphorylated form of the ubiquitously expressed serine/threonine protein kinase p-AKT, as the p-AKT inhibition prevented NCX3 upregulation. The relevant role of NCX3 during postconditioning was further confirmed by results showing that NCX3 silencing, induced by intracerebroventricular infusion of small interfering RNA (siRNA), partially reverted the postconditioning-induced neuroprotection. The results of this study support the idea that the enhancement of NCX3 expression and activity might represent a reasonable strategy to reduce the infarct extension after stroke.

NCX1 and NCX3: Two new effectors of delayed preconditioning in brain ischemia

Substantial evidence has established that a short sub-lethal brain ischemia applied before a prolonged harmful ischemic episode confers ischemic neuroprotection, a phenomenon named ischemic preconditioning. Na(+)/Ca(2+) exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, are plasmamembrane ionic transporters widely distributed in the brain, where they are involved in the control of Na(+) and Ca(2+) homeostasis and in the progression of stroke damage. The objective of this study was to evaluate the role of these three proteins in the preconditioning-induced neuroprotection. NCX protein expression was evaluated at different time points in the ischemic temporoparietal cortex of rats subjected to ischemia alone, to ischemic preconditioning alone, or to ischemic preconditioning plus ischemia. NCX1 and NCX3 were up-regulated in those brain regions protected by preconditioning treatment. These changes were mediated by p-AKT, since the p-AKT inhibition prevented the up-regulation of both isoforms. The relevant role of NCX1 and NCX3 during preconditioning was further confirmed when NCX1 and NCX3 silencing, induced by icv infusion of siRNA, partially reverted the preconditioning-induced neuroprotection. The enhancement of NCX1 and NCX3 expression and activity might represent a reasonable strategy to reduce the infarct extension after stroke.

NCX as a Key Player in the Neuroprotection Exerted by Ischemic Preconditioning and Postconditioning

Ischemic preconditioning is a neuroprotective mechanism in which a brief non-injurious episode of ischemia protects the brain from a subsequent lethal insult. Recently, it has been reported that modified reperfusion subsequent to a prolonged ischemic episode may also confer neuroprotection, a phenomenon termed postconditioning. Mitogen-activated protein kinases (MAPK) play a key role in these two neuroprotective mechanisms. The aim of this study was to evaluate whether Na(+)/Ca(2+) exchangers (NCXs), a family of ionic transporters that contribute to the maintenance of intracellular ionic homeostasis, contribute to the neuroprotection elicited by ischemic preconditioning and postconditioning.Results of this study indicated that (1) NCX1 and NCX3 are upregulated in those brain regions protected by preconditioning, while (2) postconditioning treatment induces an upregulation only in NCX3 expression. (3) NCX1 upregulation and NCX3 upregulation are mediated by p-AKT since its inhibition reverted the neuroprotective effect of preconditioning and postconditioning and prevented NCXs overexpression. (4) The involvement of NCX in preconditioning and postconditioning neuroprotection is further supported by the results of experiments showing that a partial reversion of the protective effect induced by preconditioning was obtained by silencing NCX1 or NCX3, while the silencing of NCX3 was able to mitigate the protection induced by ischemic postconditioning.Altogether, the data presented here suggest that NCX1 and NCX3 -represent two promising druggable targets for setting on new strategies in stroke therapy.

nNOS and p-ERK involvement in the neuroprotection exerted by remote postconditioning in rats subjected to transient middle cerebral artery occlusion

It has recently been hypothesized that a sub-lethal ischemic insult induced in one organ is able to protect from a harmful ischemia occurring in a different organ. The objective of this study is to identify new putative mechanisms of neuroprotection elicited by remote ischemic femoral postconditioning. A 50% reduction in the infarct volume was observed when 100min of middle cerebral artery occlusion was followed, 10min later, by the remote postconditioning stimulus represented by 20min of femoral artery occlusion. The use of in vivo silencing strategy allowed to demonstrate that NO production through nNOS mediates part of the neuroprotection. Indeed, whereas CNS nNOS expression was up-regulated by remote postconditioning, the pharmacological inhibition of nNOS or its silencing-mediated knocking-down partially prevented this neuroprotective effect. This nNOS overexpression seemed to be p-ERK dependent. In fact, p-ERK expression increased in brain cortex after remote postconditioning, and its pharmacological inhibition prevented both nNOS overexpression and remote postconditioning-mediated neuroprotection. Interestingly, neuroprotection induced by remote postconditioning was partially prevented when ganglion transmission was pharmacologically interrupted by hexamethonium, thus showing that neural factors are involved in this phenomenon. Collectively, the present study demonstrates that p-ERK and nNOS take part to the complex cascade of events triggered by ischemic remote postconditioning.

Protective Effects of Quercetin on Rat Pial Microvascular Changes during Transient Bilateral Common Carotid Artery Occlusion and Reperfusion

The aim of this study was to assess the in vivo effects of quercetin on pial microvascular responses during transient bilateral common carotid artery occlusion (BCCAO) and reperfusion. Rat pial microcirculation was visualized by fluorescence microscopy through a closed cranial window. Pial arterioles were classified in five orders of branchings. Capillaries were assigned order 0, the smallest arterioles order 1, and the largest ones order 5. In ischemic rats, 30 min BCCAO and 60 min reperfusion caused arteriolar diameter decrease (by 14.5 ± 3.3% of baseline in order 2), microvascular leakage [0.47 ± 0.04, normalized gray levels (NGL)], leukocyte adhesion in venules (9 ± 2/100 μm venular length, v.l./30 s), and reduction of capillary perfusion (by 40 ± 7% of baseline). Moreover, at the end of BCCAO and reperfusion there was a significant increase in reactive oxygen species (ROS) formation when compared with baseline. Quercetin highest dose determined dilation in all arteriolar orders (by 40 ± 4% of baseline in order 2) and prevented microvascular permeability (0.15 ± 0.02 NGL), leukocyte adhesion (3 ± 1/100 μm v.l./30 s) as well as ROS formation, while capillary perfusion was protected. Inhibition of endothelial nitric oxide synthase (NOS) prior to quercetin reduced arteriolar dilation (order 2 diameter increase by 10.3 ± 2.5% of baseline) and caused permeability increase (0.29 ± 0.03 NGL); inhibition of neuronal NOS or inducible NOS did not affect quercetin-induced effects. Inhibition of guanylyl cyclase prior to quercetin reversed the quercetin’s effects on pial arteriolar diameter and leakage. In conclusion, quercetin was able to protect pial microcirculation from ischemia–reperfusion damage inducing arteriolar dilation likely by nitric oxide release. Moreover, quercetin scavenger activity blunted ROS formation preserving the blood–brain barrier integrity.

Effects of Postconditioning on Neurogenesis and Angiogenesis During the Recovery Phase After Focal Cerebral Ischemia

Background and Purpose— Postconditioning may be a clinically feasible way to protect the brain after a stroke. However, its effects during the recovery phase post stroke remain to be fully elucidated. Here, we examine the hypothesis that ischemic postconditioning amplifies neurogenesis and angiogenesis during stroke recovery. Methods— Male Sprague–Dawley rats were subjected to 100-minute transient middle cerebral artery occlusion (MCAO) or postconditioning (100-minute middle cerebral artery occlusion plus 10-minute reperfusion plus 10-minute reocclusion). After 2 weeks, infarct volumes, behavioral outcomes, and immunohistochemical markers of neurogenesis and angiogenesis were quantified. Results— Postconditioning significantly reduced infarction and improved neurological outcomes. Concomitantly, brains subjected to postconditioning showed an increase in doublecortin/BrdU and collagen-IV/Ki67-positive cells. Conclusions— These results suggest that therapeutic effects of postconditioning may involve the promotion of neurogenesis and angiogenic remodeling during the recovery phase after focal cerebral ischemia.

Rat Pial Microvascular Responses to Transient Bilateral Common Carotid Artery Occlusion and Reperfusion: Quercetin’s Mechanism of Action

The aim of the present study was to assess quercetin’s mechanism of action in rat pial microvessels during transient bilateral common carotid artery occlusion (BCCAO) and reperfusion. Rat pial microcirculation was visualized using fluorescence microscopy through a closed cranial window. Pial arterioles were classified in five orders of branchings. In ischemic rats, 30 min BCCAO and 60 min reperfusion caused arteriolar diameter decrease, microvascular leakage, leukocyte adhesion in venules, and reduction of capillary perfusion. Quercetin highest dose determined dilation in all arteriolar orders, by 40 ± 4% of baseline in order 2 vessels, and prevented microvascular permeability [0.15 ± 0.02 normalized gray levels (NGL)], leukocyte adhesion, and capillary failure. Protein kinase C (PKC) inhibition exerted by chelerythrine prior to quercetin attenuated quercetin-induced effects: order 2 arterioles dilated by 19.0 ± 2.4% baseline, while there was an increase in permeability (0.40 ± 0.05 NGL) and leukocyte adhesion with a marked decrease in capillary perfusion. Tyrosine kinase (TK) inhibition by tyrphostin 47 prior to quercetin lessened smaller pial arterioles responses, dilating by 20.7 ± 2.5% of baseline, while leakage increased (0.39 ± 0.04 NGL) sustained by slight leukocyte adhesion and ameliorated capillary perfusion. Inhibition of endothelium nitric oxide synthase (eNOS) by NG-nitro-L-arginine-methyl ester (L-NAME) prior to PKC or TK reduced the quercetin’s effects on pial arteriolar diameter and leakage. eNOS inhibition by L-NAME reduced quercetin effects on pial arteriolar diameter and leakage. Finally, combined inhibition of PKC and TK prior to quercetin abolished quercetin-induced effects, decreasing eNOS expression, while blocking ATP-sensitive potassium (KATP) channels by glibenclamide suppressed arteriolar dilation. In conclusion, the protective effects of quercetin could be due to different mechanisms resulting in NO release throughout PKC and TK intracellular signaling pathway activation.

Anesthesia and Surgery Impair Blood–Brain Barrier and Cognitive Function in Mice

Blood–brain barrier (BBB) dysfunction, e.g., increase in BBB permeability, has been reported to contribute to cognitive impairment. However, the effects of anesthesia and surgery on BBB permeability, the underlying mechanisms, and associated cognitive function remain largely to be determined. Here, we assessed the effects of surgery (laparotomy) under 1.4% isoflurane anesthesia (anesthesia/surgery) for 2 h on BBB permeability, levels of junction proteins and cognitive function in both 9- and 18-month-old wild-type mice and 9-month-old interleukin (IL)-6 knockout mice. BBB permeability was determined by dextran tracer (immunohistochemistry imaging and spectrophotometric quantification), and protein levels were measured by Western blot and cognitive function was assessed by using both Morris water maze and Barnes maze. We found that the anesthesia/surgery increased mouse BBB permeability to 10-kDa dextran, but not to 70-kDa dextran, in an IL-6-dependent and age-associated manner. In addition, the anesthesia/surgery induced an age-associated increase in blood IL-6 level. Cognitive impairment was detected in 18-month-old, but not 9-month-old, mice after the anesthesia/surgery. Finally, the anesthesia/surgery decreased the levels of β-catenin and tight junction protein claudin, occludin and ZO-1, but not adherent junction protein VE-cadherin, E-cadherin, and p120-catenin. These data demonstrate that we have established a system to study the effects of perioperative factors, including anesthesia and surgery, on BBB and cognitive function. The results suggest that the anesthesia/surgery might induce an age-associated BBB dysfunction and cognitive impairment in mice. These findings would promote mechanistic studies of postoperative cognitive impairment, including postoperative delirium.