CHARACTERIZATION OF A MOUSE MODEL OF HYPOXIC PRECONDITIONING IN NEONATAL HYPOXIA ISCHEMIA

BACKGROUND: Hypoxic-ischemic (HI) damage in neonatal brain is a major risk factor for different human disorders. So far, no drug is presently available to manage this clinical emergency, therefore, the attention of researchers working in the field has been given to endogenous neuroprotective strategies, in order to identify new druggable targets. In particular, one of the best endogenous neuroprotective strategies is Preconditioning (PC), which is a subliminal stimulus able to protect the brain from a subsequent harmful stimulus. AIM: The first aim of the present work was to identify a new model of hypoxic preconditioning. Then, we investigated whether hypoxic preconditioning (HPC) is able to stimulate the differentiation of neural stem cells (NSCs) in a mouse model of neonatal hypoxia ischemia. In addition, we investigated the relationship between the alteration of ionic homeostasis and the activation of endogenous neurogenesis mediated by NCX. The plasma membrane protein Na+/Ca2+ exchanger (NCX), whose activity has been linked to brain ischemic pathophysiology in adult animals. METHODS: Seven-day-old C57BL/6 mice were divided into different experimental groups. Hypoxia ischemia was induced by using Rice-Vannucci model. Briefly, they were subjected to ligation and cutting of the right common carotid artery followed by an exposure to hypoxia (92% N2 and 8% O2) at different time intervals and sacrificed at different times of reperfusion. Histopathological damage in the hippocampus was determined by measuring the expression level of Propidium Iodide (PI), whereas the neurogenesis was evaluated by using immunohistochemistry with different markers. The development of sensorimotor reflexes was examined by behavioral tests. The increase of NCX expression was evaluated by using immunohistochemical analyses in hippocampus dentate gyrus. RESULTS: As expected, the greatest damage was found in mice subjected to ischemia plus 60’ hypoxia (HI 60’) and sacrificed 7 days after ischemia induction. A significant reduction in the hippocampal damage was observed in mice subjected to hypoxia for 20 minutes at postnatal day seven (P7) followed, 3 days later, by HI 60’ (P10) and sacrificed at postnatal day eleven (P11). Indeed, this result suggests that 20’ hypoxia functions as a preconditioning stimulus. In animals subjected to hypoxic-ischemic insult the damage was mainly localized in CA1 and CA3 regions, whereas the dentate gyrus was spared. Concerning the mechanism by which preconditioning may exert its effects, we found that preconditioning stimulus was able to trigger an increased expression of Nestin, a marker of neurogenesis, in seven-day-old mice. In addition, hypoxic preconditioning was able to determine the increase of proliferating cells (BrdU+/PSANCAM+) and neuroblast cells (PSANCAM+) in the dentate gyrus of HI 60’ (P10) mice, sacrificed at P11. In subventricular zone hypoxic preconditioning determined the increase of neuroblast cells (PSANCAM+). Moreover, we observed an increase of NCX1 and NCX3 positive cells in dentate gyrus of preconditioned hypoxic-ischemic animals. Interestingly, the preconditioned hypoxic-ischemic animals also showed a better performance in the cliff avoidance and geotaxis reflex test than HI 60’ mice. CONCLUSIONS: Our results indicate that hypoxic preconditioning is associated to endogenous neurogenesis in immature brain after insult. The increase of neurogenic process is probably correlated to ionic homeostasis maintenance, regulated by NCX proteins. Therefore, NCX proteins can represent potential pharmacological targets for the treatment of brain damage associated to neonatal hypoxic-ischemic encephalopathy (HIE)

Models and methods for conditioning the ischemic brain

Abstract Background In the last decades the need to find new neuroprotective targets has addressed the researchers to investigate the endogenous molecular mechanisms that brain activates when exposed to a conditioning stimulus. Indeed, conditioning is an adaptive biological process activated by those interventions able to confer resistance to a deleterious brain event through the exposure to a sub-threshold insult. Specifically, preconditioning and postconditioning are realized when the conditioning stimulus is applied before or after, respectively, the harmul ischemia. Aims and Results The present review will describe the most common methods to induce brain conditioning, with particular regards to surgical, physical exercise, temperature-induced and pharmacological approaches. It has been well recognized that when the subliminal stimulus is delivered after the ischemic insult, the achieved neuroprotection is comparable to that observed in models of ischemic preconditioning. In addition, subjecting the brain to both preconditioning as well as postconditioning did not cause greater protection than each treatment alone. Conclusions The last decades have provided fascinating insights into the mechanisms and potential application of strategies to induce brain conditioning. Since the identification of intrinsic cell‐survival pathways should provide more direct opportunities for translational neuroprotection trials, an accurate examination of the different models of preconditioning and postconditioning is mandatory before starting any new project

Ionic homeostasis in brain conditioning

Most of the current focus on developing neuroprotective therapies is aimed at preventing neuronal death. However, these approaches have not been successful despite many years of clinical trials mainly because the numerous side effects observed in humans and absent in animals used at preclinical level. Recently, the research in this field aims to overcome this problem by developing strategies which induce, mimic, or boost endogenous protective responses and thus do not interfere with physiological neurotransmission. Preconditioning is a protective strategy in which a subliminal stimulus is applied before a subsequent harmful stimulus, thus inducing a state of tolerance in which the injury inflicted by the challenge is mitigated. Tolerance may be observed in ischemia, seizure, and infection. Since it requires protein synthesis, it confers delayed and temporary neuroprotection, taking hours to develop, with a pick at 1-3 days. A new promising approach for neuroprotection derives from post-conditioning, in which neuroprotection is achieved by a modified reperfusion subsequent to a prolonged ischemic episode. Many pathways have been proposed as plausible mechanisms to explain the neuroprotection offered by preconditioning and post-conditioning. Although the mechanisms through which these two endogenous protective strategies exert their effects are not yet fully understood, recent evidence highlights that the maintenance of ionic homeostasis plays a key role in propagating these neuroprotective phenomena. The present article will review the role of protein transporters and ionic channels involved in the control of ionic homeostasis in the neuroprotective effect of ischemic preconditioning and post-conditioning in adult brain, with particular regards to the Na(+)/Ca2(+) exchangers (NCX), the plasma membrane Ca2(+)-ATPase (PMCA), the Na(+)/H(+) exchange (NHE), the Na(+)/K(+)/2Cl(-) cotransport (NKCC) and the acid-sensing cation channels (ASIC). Ischemic stroke is the third leading cause of death and disability. Up until now, all clinical trials testing potential stroke neuroprotectants failed. For this reason attention of researchers has been focusing on the identification of brain endogenous neuroprotective mechanisms activated after cerebral ischemia. In this context, ischemic preconditioning and ischemic post-conditioning represent two neuroprotecive strategies to investigate in order to identify new molecular target to reduce the ischemic damage

Role of IL-6/STAT3 Axis in Resistance to Cisplatin in Gastric Cancers

Gastric cancer, the second most common cause of death worldwide, is characterized by poor prognosis and low responsiveness to chemotherapy. Indeed, multidrug resistance, based mainly on cellular and molecular factors, remains one of the most limiting factors of the current approach to gastric cancer (GC) therapy. We employed a comprehensive gene expression analysis through data mining of publicly available databases to assess the role of the signal transducer and activator of transcription 3 (STAT3) in gastric cancer drug efficiency. It has been proposed that gastric cancer cells are less sensitive to these drugs because they develop resistance to these agents through activating alternative signalling pathways responsible for overcoming pharmacological inhibition. Our study evaluated the hypothesis that activating STAT3 signalling in response to cisplatin reduces the reaction to the drug. Consistent with this hypothesis, inhibition of interleukin 6 (IL-6)/STAT3 in combination therapy with cisplatin prevented both STAT3 activation and more lethality than induction by a single agent. The data suggest that the IL-6/STAT3 axis block associated with cisplatin treatment may represent a strategy to overcome resistance

Stroke by inducing HDAC9-dependent deacetylation of HIF-1 and Sp1, promotes TfR1 transcription and GPX4 reduction, thus determining ferroptotic neuronal death

: Background: The inhibition of histone deacetylase 9 (HDAC9) represents a promising druggable target for stroke intervention. Indeed, HDAC9 is overexpressed in neurons after brain ischemia where exerts a neurodetrimental role. However, mechanisms of HDAC9-dependent neuronal cell death are not yet well established. Methods: Brain ischemia was obtained in vitro by primary cortical neurons exposed to glucose deprivation plus reoxygenation (OGD/Rx) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcript and protein levels. Chromatin immunoprecipitation was used to evaluate the binding of transcription factors to the promoter of target genes. Cell viability was measured by MTT and LDH assays. Ferroptosis was evaluated by iron overload and 4-hydroxynonenal (4-HNE) release. Results: Our results showed that HDAC9 binds to hypoxia-inducible factor 1 (HIF-1) and specificity protein 1 (Sp1), two transcription activators of transferrin 1 receptor (TfR1) and glutathione peroxidase 4 (GPX4) genes, respectively, in neuronal cells exposed to OGD/Rx. Consequently, HDAC9 induced: (1) an increase in protein level of HIF-1 by deacetylation and deubiquitination, thus promoting the transcription of the pro-ferroptotic TfR1 gene; and (2) a reduction in Sp1 protein levels by deacetylation and ubiquitination, thus resulting in a down-regulation of the anti-ferroptotic GPX4 gene. Supporting these results, the silencing of HDAC9 partially prevented either HIF-1 increase and Sp1 reduction after OGD/Rx. Interestingly, silencing of the neurodetrimental factors, HDAC9, HIF-1, or TfR1 or the overexpression of the prosurvival factors Sp1 or GPX4 significantly reduced a well-known marker of ferroptosis 4-HNE after OGD/Rx. More important, in vivo, intracerebroventricular injection of siHDAC9 reduced 4-HNE levels after stroke by preventing: (1) HIF-1 and TfR1 increase and thus the augmented intracellular iron overload; and (2) a reduction of Sp1 and its target gene GPX4. Conclusions: Collectively, results obtained suggest that HDAC9 mediates post-traslational modifications of HIF-1 and Sp1 that, in turn, increases TfR1 and decreases GPX4 expression, thus promoting neuronal ferroptosis in in vitro and in vivo models of stroke

Smoking cessation in patients requiring bronchoscopy: The Bronchoscopy AntiSmoking Study (BASIS)

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Ruta graveolens water extract (RGWE) ameliorates ischemic damage and improves neurological deficits in a rat model of transient focal brain ischemia

The limited therapeutic options for ischemic stroke treatment render necessary the identification of new strategies. In recent years, it has been shown that natural compounds may represent a valid therapeutic opportunity. Therefore, the present study aimed to evaluate the protective effect of Ruta graveolens water extract (RGWE) in an in vivo experimental model of brain ischemia

Methylmercury upregulates RE-1 silencing transcription factor (REST) in SH-SY5Y cells and mouse cerebellum

Methylmercury (MeHg) is a highly neurotoxic compound that, in adequate doses, can cause damage to the brain, including developmental defects and in severe cases cell death. The RE-1-silencing transcription factor (REST) has been found to be involved in the neurotoxic effects of environmental pollutants such as polychlorinated biphenyls (PCBs). In this study, we investigated the effects of MeHg treatment on REST expression and its role in MeHg-induced neurotoxicity in neuroblastoma SH-SY5Y cells. We found that MeHg exposure caused a dose- and time- dependent apoptotic cell death, as evidenced by the appearance of apoptotic hallmarks including caspase-3 processing and annexin V uptake. Moreover, MeHg increased REST gene and gene product expression. MeHg-induced apoptotic cell death was completely abolished by REST knockdown. Interestingly, MeHg (1. μM/24. h) increased the expression of REST Corepressor (Co-REST) and its binding with REST whereas the other REST cofactor mammalian SIN3 homolog A transcription regulator (mSin3A) was not modified. In addition, we demonstrated that the acetylation of histone protein H4 was reduced after MeHg treatment and was critical for MeHg-induced apoptosis. Accordingly, the pan-histone deacetylase inhibitor trichostatin-A (TSA) prevented MeHg-induced histone protein H4 deacetylation, thereby reverting MeHg-induced neurotoxic effect. Male mice subcutaneously injected with 10 mg/kg of MeHg for 10 days showed an increase in REST expression in the granule cell layer of the cerebellum together with a decrease in histone H4 acetylation. Collectively, we demonstrated that methylmercury exposure can cause neurotoxicity by activating REST gene expression and H4 deacetylation

Prolonged NCX activation prevents SOD1 accumulation, reduces neuroinflammation, ameliorates motor behavior and prolongs survival in a ALS mouse model.

Abstract Imbalance in cellular ionic homeostasis is a hallmark of several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS). Sodium-calcium exchanger (NCX) is a membrane antiporter that, operating in a bidirectional way, couples the exchange of Ca2+ and Na + ions in neurons and glial cells, thus controlling the intracellular homeostasis of these ions. Among the three NCX genes, NCX1 and NCX2 are widely expressed within the CNS, while NCX3 is present only in skeletal muscles and at lower levels of expression in selected brain regions. ALS mice showed a reduction in the expression and activity of NCX1 and NCX2 consistent with disease progression, therefore we aimed to investigate their role in ALS pathophysiology. Notably, we demonstrated that the pharmacological activation of NCX1 and NCX2 by the prolonged treatment of SOD1G93A mice with the newly synthesized compound neurounina: (1) prevented the reduction in NCX activity observed in spinal cord; (2) preserved motor neurons survival in the ventral spinal horn of SOD1G93A mice; (3) prevented the spinal cord accumulation of misfolded SOD1; (4) reduced astroglia and microglia activation and spared the resident microglia cells in the spinal cord; (5) improved the lifespan and mitigated motor symptoms of ALS mice. The present study highlights the significant role of NCX1 and NCX2 in the pathophysiology of this neurodegenerative disorder and paves the way for the design of a new pharmacological approach for ALS