RESUMEN
Efficient cell-to-cell communication is essential for tissue development, homeostasis, and the maintenance of cellular functions after injury. Tunneling nanotubes (TNTs) have emerged as a new important method of cell-to-cell communication. TNTs are primarily established between stressed and unstressed cells and can transport a variety of cellular components. Mitochondria are important trafficked entities through TNTs. Transcellular mitochondria transfer permits the incorporation of healthy mitochondria into the endogenous network of recipient cells, changing the bioenergetic profile and other functional properties of the recipient and may allow the recipient cells to recuperate from apoptotic processes and return to a normal operating state. Mesenchymal cells (MSCs) can form TNTs and transfer mitochondria and other constituents to target cells. This occurs under both physiological and pathological conditions, leading to changes in cellular energy metabolism and functions. This review summarizes the newly described capacity of melatonin to improve mitochondrial fusion/fission dynamics and promote TNT formation. This new evidence suggests that melatonin's protective effects could be attributed to its ability to prevent mitochondrial damage in injured cells, reduce senescence, and promote anastasis, a natural cell recovery phenomenon that rescues cells from the brink of death. The modulation of these new routes of intercellular communication by melatonin could play a key role in increasing the therapeutic potential of MSCs.
Asunto(s)
Melatonina , Células Madre Mesenquimatosas , Nanotubos , Comunicación Celular/fisiología , Estructuras de la Membrana Celular , Melatonina/metabolismo , Melatonina/farmacología , Células Madre Mesenquimatosas/metabolismoRESUMEN
Neonatal encephalopathy (NE) is a pathological condition affecting long-term neurodevelopmental outcomes. Hypothermia is the only therapeutic option, but does not always improve outcomes; hence, researchers continue to hunt for pharmaceutical compounds. Melatonin treatment has benefitted neonates with hypoxic-ischemic (HI) brain injury. However, unlike animal models that enable the study of the brain and the pathophysiologic cascade, only blood is available from human subjects. Therefore, due to the unavailability of neonatal brain tissue, assumptions about the pathophysiology in pathways and cascades are made in human subjects with NE. We analyzed animal and human specimens to improve our understanding of the pathophysiology in human neonates. A neonate with NE who underwent hypothermia and enrolled in a melatonin pharmacokinetic study was compared to HI rats treated/untreated with melatonin. MicroRNA (miRNA) analyses provided profiles of the neonate's plasma, rat plasma, and rat brain cortexes. We compared these profiles through a bioinformatics tool, identifying Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways common to HI brain injury and melatonin treatment. After evaluating the resulting pathways and the literature, to validate the method, the key proteins expressed in HI brain injury were investigated using cerebral cortexes. The upregulated miRNAs in human neonate and rat plasma helped identify two KEGG pathways, glioma and long-term potentiation, common to HI injury and melatonin treatment. A unified neonatal cerebral melatonin-sensitive HI pathway was designed and validated by assessing the expression of protein kinase Cα (PKCα), phospho (p)-Akt, and p-ERK proteins in rat brain cortexes. PKCα increased in HI-injured rats and further increased with melatonin. p-Akt and p-ERK returned phosphorylated to their basal level with melatonin treatment after HI injury. The bioinformatics analyses validated by key protein expression identified pathways common to HI brain injury and melatonin treatment. This approach helped complete pathways in neonates with NE by integrating information from animal models of HI brain injury.
Asunto(s)
Lesiones Encefálicas , Hipotermia , Hipoxia-Isquemia Encefálica , Melatonina , MicroARNs , Animales , Animales Recién Nacidos , Humanos , Hipotermia/tratamiento farmacológico , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Hipoxia-Isquemia Encefálica/genética , Hipoxia-Isquemia Encefálica/metabolismo , Melatonina/farmacología , Melatonina/uso terapéutico , MicroARNs/genética , Proteína Quinasa C-alfa , Proteínas Proto-Oncogénicas c-akt , RatasRESUMEN
Mitochondrial dysfunction is considered one of the hallmarks of ischemia/reperfusion injury. Mitochondria are plastic organelles that undergo continuous biogenesis, fusion, and fission. They can be transferred between cells through tunneling nanotubes (TNTs), dynamic structures that allow the exchange of proteins, soluble molecules, and organelles. Maintaining mitochondrial dynamics is crucial to cell function and survival. The present study aimed to assess the effects of melatonin on mitochondrial dynamics, TNT formation, and mitochondria transfer in HT22 cells exposed to oxygen/glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin treatment during the reoxygenation phase reduced mitochondrial reactive oxygen species (ROS) production, improved cell viability, and increased the expression of PGC1α and SIRT3. Melatonin also preserved the expression of the membrane translocase proteins TOM20 and TIM23, and of the matrix protein HSP60, which are involved in mitochondrial biogenesis. Moreover, it promoted mitochondrial fusion and enhanced the expression of MFN2 and OPA1. Remarkably, melatonin also fostered mitochondrial transfer between injured HT22 cells through TNT connections. These results provide new insights into the effect of melatonin on mitochondrial network reshaping and cell survival. Fostering TNTs formation represents a novel mechanism mediating the protective effect of melatonin in ischemia/reperfusion injury.
Asunto(s)
Isquemia Encefálica/patología , Estructuras de la Membrana Celular/efectos de los fármacos , Melatonina/farmacología , Mitocondrias/efectos de los fármacos , Neuronas/ultraestructura , Animales , Línea Celular , Hipocampo/efectos de los fármacos , Hipocampo/patología , Hipocampo/ultraestructura , Ratones , Mitocondrias/metabolismo , Nanotubos , Neuronas/efectos de los fármacos , Neuronas/patología , Daño por Reperfusión/patologíaRESUMEN
INTRODUCTION: Neonates with hypoxic-ischemic encephalopathy (HIE) undergoing hypothermia may benefit from adjunctive therapy with melatonin. However, melatonin safety, pharmacokinetics (PK), and dosage in this sensitive population are still unknown. METHODS AND RESULTS: This study assessed the PK and safety of melatonin enteral administration to neonates with HIE undergoing hypothermia. Melatonin was infused at 0.5 mg/kg in five neonates with HIE undergoing hypothermia. Infusion started 1 hour after the neonates reached the target temperature of 33.5°C. Blood samples were collected before and at selective times after melatonin infusion. Abdominal complications or clinically significant changes in patients' vital signs were not found during or after melatonin. The peak plasma concentration reached 0.25 µg/mL. The area under the curve in 24 hours was 4.35 µg/mL*h. DISCUSSION: Melatonin half-life and clearance were prolonged, and the distribution volume decreased compared to adults. In silico simulation estimated that the steady state can be reached after four infusions. Hypothermia does not affect melatonin PK. In humans high blood concentrations with lower doses can be achieved compared to animal experimentation, although intravenous administration is advised in the neonate population. Our study is a preparatory step for future clinical studies aimed at assessing melatonin efficacy in HIE.
Asunto(s)
Hipotermia Inducida , Melatonina/farmacocinética , Femenino , Humanos , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/terapia , Recién Nacido , Masculino , Melatonina/uso terapéuticoRESUMEN
Increasing evidence indicates that melatonin possesses protective effects toward different kinds of damage in various organs, including the brain. In a neonatal model of hypoxia-ischemia (HI), melatonin was neuroprotective and preserved the expression of the silent information regulator 1 (SIRT1) 24 hours after the insult. This study aimed to gain more insight into the role of SIRT1 in the protective effect of melatonin after HI by studying the early (1 hour) modulation of SIRT1 and its downstream targets, and the consequences on necrosis, apoptosis, autophagy, and glial cell activation. We found that melatonin administered 5 minutes after the ischemic insult significantly reduced necrotic cell death assessed 1 hour after its administration. In parallel, we found a reduced activation of the early phases of intrinsic apoptosis, detected by reduced BAX translocation to the mitochondria and preservation of the mitochondrial expression of cytochrome C, indicating a reduced outer mitochondrial membrane permeabilization in the melatonin-treated ischemic animals. These effects were concomitant to increased expression and activity of SIRT1, reduced expression and acetylation of p53, and increased autophagy activation. Melatonin also reduced HI-induced glial cells activation. SIRT1 was expressed in neurons after HI and melatonin but not in reactive glial cells expressing GFAP. Colocalization between SIRT1 and GFAP was found in some cells in control conditions. In summary, our results provide more insight into the connection between SIRT1 and melatonin in neuroprotection. The possibility that melatonin-induced SIRT1 activity might contribute to differentiate neuronal progenitor cells during the neurodegenerative process needs to be further investigated.
Asunto(s)
Isquemia Encefálica/metabolismo , Melatonina/metabolismo , Sirtuina 1/metabolismo , Animales , Animales Recién Nacidos , Isquemia Encefálica/patología , Muerte Celular , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Neuronas/metabolismo , Embarazo , Ratas Sprague-Dawley , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Melatonin possesses potential efficacy in perinatal brain injuries, and has been proposed as adjunctive pharmacological therapy in combination with hypothermia in the clinical setting. However, the pharmacokinetics of melatonin in preterm and term newborns is still unknown. The aim of this study was to analyze the pharmacokinetics of melatonin after intragastric administration in preterm infants. Preterm newborns were enrolled 24-72 h after birth, and randomly assigned to three groups receiving a single bolus of 0.5 mg·kg-1 melatonin, or 3 boluses of 1 or 5 mg·kg-1 of melatonin at 24-h intervals. Blood samples were collected before and at selective times after melatonin administration. The half-life of melatonin in plasma ranged from 7.98 to 10.94 h, and the area under the curve (AUC) from 10.48 to 118.17 µg·mL-1·h-1. Our results indicate a different pharmacokinetic profile in premature newborns, compared to adults and experimental animals. The high peak plasma concentrations and the long half-life indicate that in the neonatal clinical setting, it is possible to obtain and maintain high serum concentrations using a single administration of melatonin repeated every 12/24 h.
Asunto(s)
Melatonina/farmacocinética , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacocinética , Administración Oral , Adulto , Área Bajo la Curva , Terapia Combinada , Relación Dosis-Respuesta a Droga , Femenino , Semivida , Humanos , Lactante , Recién Nacido , Recien Nacido Prematuro , Melatonina/administración & dosificación , Melatonina/sangre , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/sangre , EmbarazoRESUMEN
We herein report the results from a comparative study of arsenite toxicity in respiration-proficient (RP) and -deficient (RD) U937 cells. An initial characterization of these cells led to the demonstration that the respiration-deficient phenotype is not associated with apparent changes in mitochondrial mass and membrane potential. In addition, similar levels of superoxide (O2(.-)) were generated by RP and RD cells in response to stimuli specifically triggering respiratory chain-independent mitochondrial mechanisms or extramitochondrial, NADPH-oxidase dependent, mechanisms. At the concentration of 2.5µM, arsenite elicited selective formation of O2(.-) in the respiratory chain of RP cells, with hardly any contribution of the above mechanisms. Under these conditions, O2(.-) triggered downstream events leading to endoplasmic reticulum (ER) stress, autophagy and apoptosis. RD cells challenged with similar levels of arsenite failed to generate O2(.-) because of the lack of a functional respiratory chain and were therefore resistant to the toxic effects mediated by the metalloid. Their resistance, however, was lost after exposure to four fold greater concentrations of arsenite, coincidentally with the release of O2(.-) mediated by NADPH oxidase. Interestingly, extramitochondrial O2(.-) triggered the same downstream events and an identical mode of death previously observed in RP cells. Taken together, the results obtained in this study indicate that arsenite toxicity is strictly dependent on O2(.-) availability that, regardless of whether generated in the mitochondrial or extramitochondrial compartments, triggers similar downstream events leading to ER stress, autophagy and apoptosis.
Asunto(s)
Arsenitos/toxicidad , Respiración de la Célula , NADPH Oxidasas/metabolismo , Superóxidos/metabolismo , Apoptosis/efectos de los fármacos , Autofagia , Supervivencia Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Consumo de Oxígeno , Células U937RESUMEN
Maternal infection/inflammation represents one of the most important factors involved in the etiology of brain injury in newborns. We investigated the modulating effect of prenatal melatonin on the neonatal brain inflammation process resulting from maternal intraperitoneal (i.p.) lipopolysaccharide (LPS) injections. LPS (300 µg/kg) was administered to pregnant rats at gestational days 19 and 20. Melatonin (5 mg/kg) was administered i.p. at the same time as LPS. Melatonin counteracted the LPS sensitization to a second ibotenate-induced excitotoxic insult performed on postnatal day (PND) 4. As melatonin succeeded in reducing microglial activation in neonatal brain at PND1, pathways previously implicated in brain inflammation regulation, such as endoplasmic reticulum (ER) stress, autophagy and silent information regulator 1 (SIRT1), a melatonin target, were assessed at the same time-point in our experimental groups. Results showed that maternal LPS administrations resulted in an increase in CHOP and Hsp70 protein expression and eIF2α phosphorylation, indicative of activation of the unfolded protein response consequent to ER stress, and a slighter decrease in the autophagy process, determined by reduced lipidated LC3 and increased p62 expression. LPS-induced inflammation also reduced brain SIRT1 expression and affected the expression of miR-34a, miR146a, and miR-126. All these effects were blocked by melatonin. Cleaved-caspase-3 apoptosis pathway did not seem to be implicated in the noxious effect of LPS on the PND1 brain. We conclude that melatonin is effective in reducing maternal LPS-induced neonatal inflammation and related brain injury. Its role as a prophylactic/therapeutic drug deserves to be investigated by clinical studies.
Asunto(s)
Autofagia/efectos de los fármacos , Encéfalo/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Melatonina/farmacología , MicroARNs/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Animales Recién Nacidos , Encéfalo/patología , Femenino , Inflamación/metabolismo , Inflamación/patología , Lipopolisacáridos/toxicidad , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Efectos Tardíos de la Exposición Prenatal/patología , Ratas , Ratas WistarRESUMEN
Conditions that interfere with the endoplasmic reticulum (ER) functions cause accumulation of unfolded proteins in the ER lumen, referred to as ER stress, and activate a homeostatic signaling network known as unfolded protein response (UPR). We have previously shown that in neonatal rats subjected to hypoxia-ischemia (HI), melatonin administration significantly reduces brain damage. This study assessed whether attenuation of ER stress is involved in the neuroprotective effect of melatonin after neonatal HI. We found that the UPR was strongly activated after HI. Melatonin significantly reduced the neuron splicing of XBP-1 mRNA, the increased phosphorylation of eIF2α, and elevated expression of chaperone proteins GRP78 and Hsp70 observed after HI in the brain. CHOP, which plays a convergent role in the UPR, was reduced as well. Melatonin also completely prevented the depletion of SIRT-1 induced by HI, and this effect was observed in the same neurons that over-express CHOP. These results demonstrate that melatonin reduces ER stress induced by neonatal HI and preserves SIRT-1 expression, suggesting that SIRT-1, due to its action in the modulation of a wide variety of signaling pathways involved in neuroprotection, may play a key role in the reduction of ER stress and neuroprotection observed after melatonin.
Asunto(s)
Estrés del Retículo Endoplásmico/efectos de los fármacos , Hipoxia-Isquemia Encefálica/metabolismo , Melatonina/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Sirtuina 1/metabolismo , Animales , Animales Recién Nacidos , RatasRESUMEN
Promoting neural cell proliferation may represent an important strategy for enhancing brain repair after developmental brain injury. The present study aimed to assess the effects of melatonin on cell proliferation after an ischemic injury in the developing hippocampus, focusing on cell cycle dynamics. After in vivo neonatal hypoxia-ischemia (HI), hippocampal cell cycle dynamics were assessed by flow cytometry, together with histological evaluation of dentate gyrus cellularity and proliferation. Melatonin significantly increased the number of proliferating cells in the G2/M phase as well as the proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) labeling reduced by HI. In vivo BrdU labeling revealed a higher BrdU-positivity in the dentate gyrus of ischemic rats treated with melatonin, an effect followed by increased cellularity and preserved hippocampal tissue integrity. These results indicate that the protective effect of melatonin after ischemic injury in neonatal rats may rely on the modulation of cell cycle dynamics of newborn hippocampal cells and increased cell proliferation.
Asunto(s)
Animales Recién Nacidos , Ciclo Celular , Proliferación Celular , Proteína Doblecortina , Hipocampo , Melatonina , Animales , Melatonina/farmacología , Melatonina/uso terapéutico , Hipocampo/patología , Hipocampo/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ratas Wistar , Hipoxia-Isquemia Encefálica/patología , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Ratas , MasculinoRESUMEN
Mitochondria functionally degrade as neurons age. Degenerative changes cause inefficient oxidative phosphorylation (OXPHOS) and elevated electron leakage from the electron transport chain (ETC) promoting increased intramitochondrial generation of damaging reactive oxygen and reactive nitrogen species (ROS and RNS). The associated progressive accumulation of molecular damage causes an increasingly rapid decline in mitochondrial physiology contributing to aging. Melatonin, a multifunctional free radical scavenger and indirect antioxidant, is synthesized in the mitochondrial matrix of neurons. Melatonin reduces electron leakage from the ETC and elevates ATP production; it also detoxifies ROS/RNS and via the SIRT3/FOXO pathway it upregulates activities of superoxide dismutase 2 and glutathione peroxidase. Melatonin also influences glucose processing by neurons. In neurogenerative diseases, neurons often adopt Warburg-type metabolism which excludes pyruvate from the mitochondria causing reduced intramitochondrial acetyl coenzyme A production. Acetyl coenzyme A supports the citric acid cycle and OXPHOS. Additionally, acetyl coenzyme A is a required co-substrate for arylalkylamine-N-acetyl transferase, which rate limits melatonin synthesis; therefore, melatonin production is diminished in cells that experience Warburg-type metabolism making mitochondria more vulnerable to oxidative stress. Moreover, endogenously produced melatonin diminishes during aging, further increasing oxidative damage to mitochondrial components. More normal mitochondrial physiology is preserved in aging neurons with melatonin supplementation.
RESUMEN
Mesenchymal stem cells are multipotent stem cells that reside in many human tissues and organs. Mesenchymal stem cells are widely used in experimental and clinical regenerative medicine due to their capability to transdifferentiate into various lineages. However, when transplanted, they lose part of their multipotency and immunomodulatory properties, and most of them die after injection into the damaged tissue. In this review, we discuss the potential utility of melatonin in preserving mesenchymal stem cells' survival and function after transplantation. Melatonin is a pleiotropic molecule regulating critical cell functions including apoptosis, endoplasmic reticulum stress, and autophagy. Melatonin is also synthesized in the mitochondria where it reduces oxidative stress, the opening of the mitochondrial permeability transition pore and the downstream caspase activation, activates uncoupling proteins, and curtails the proinflammatory response. In addition, recent findings showed that melatonin also promotes the formation of tunneling nanotubes and the transfer of mitochondria between cells through the connecting tubules. As mitochondrial dysfunction is a primary cause of mesenchymal stem cells death and senescence and a critical issue for survival after transplantation, we propose that melatonin by favoring mitochondria functionality and their transfer through tunneling nanotubes from healthy to suffering cells could improve mesenchymal stem cell-based therapy in a large number of diseases for which basic and clinical trials are underway.
RESUMEN
Despite advances in obstetric and neonatal care, challenges remain in early identification of neonates with encephalopathy due to hypoxia-ischemia who are undergoing therapeutic hypothermia. Therefore, there is a deep search for biomarkers that can identify brain injury. The aims of this study were to investigate the serum and brain expressions of two potential biomarkers, miR-126/miR-146a, in a preclinical model of hypoxia-ischemia (HI)-induced brain injury, and to explore their modulation during melatonin treatment. Seven-day-old rats were subjected to permanent ligation of the right carotid artery followed by 2.5 h hypoxia (HI). Melatonin (15 mg/kg) was administered 5 min after HI. Serum and brain samples were collected 1, 6 and 24 h after HI. Results show that HI caused a significant increase in the circulating levels of both miR-126 and miR-146a during the early phase of ischemic brain damage development (i.e. 1 h), with a parallel and opposite pattern in the ischemic cerebral cortex. These effects are not observed 24 h later. Treatment with melatonin restored the HI-induced effects on miR-126/miR-146a expressions, both in the cerebral cortex and in serum. We conclude that miR-126/miR-146a are promising biomarkers of HI injury and demonstrate an associated change in concentration following melatonin treatment.
Asunto(s)
Lesiones Encefálicas , Hipoxia-Isquemia Encefálica , Melatonina , MicroARNs , Femenino , Embarazo , Animales , Ratas , Melatonina/uso terapéutico , Animales Recién Nacidos , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Encéfalo/metabolismo , Lesiones Encefálicas/metabolismo , Biomarcadores/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Isquemia/tratamiento farmacológico , Isquemia/metabolismoRESUMEN
Surgery is frequently associated with excessive oxidative stress. Melatonin acts as an antioxidant and transient melatonin deficiency has been described in neonatal surgical patients. This randomized, blinded, prospective pilot study tested the hypothesis that oral melatonin supplementation in newborn infants undergoing surgery is effective in reducing perioperative oxidative stress. A total of twenty-three newborn infants requiring surgery were enrolled: 10 received a single dose of oral melatonin 0.5 mg/kg in the morning, before surgery (MEL group), and 13 newborns served as the control group (untreated group). Plasma concentrations of melatonin, Non-Protein-Bound Iron (NPBI), Advanced Oxidation Protein Products (AOPP), and F2-Isoprostanes (F2-IsoPs) were measured. Both in the pre- and postoperative period, melatonin concentrations were significantly higher in the MEL group than in the untreated group (preoperative: 1265.50 ± 717.03 vs. 23.23 ± 17.71 pg/mL, p < 0.0001; postoperative: 1465.20 ± 538.38 vs. 56.47 ± 37.18 pg/mL, p < 0.0001). Melatonin significantly increased from the pre- to postoperative period in the untreated group (23.23 ± 17.71 vs. 56.47 ± 37.18 pg/mL; pg/mL p = 0.006). In the MEL group, the mean blood concentrations of NPBI, F2-IsoPs, and AOPP significantly decreased from the pre- to the postoperative period (4.69 ± 3.85 vs. 1.65 ± 1.18 micromol/dL, p = 0.049; 128.40 ± 92.30 vs. 50.25 ± 47.47 pg/mL, p = 0.037 and 65.18 ± 15.50 vs. 43.98 ± 17.92 micromol/dL, p = 0.022, respectively). Melatonin concentration increases physiologically from the pre- to the postoperative period, suggesting a defensive physiologic response to counteract oxidative stress. The administration of exogenous melatonin in newborn infants undergoing surgery reduces lipid and protein peroxidation in the postoperative period, showing a potential role in protecting babies from the deleterious consequences of oxidative stress.
RESUMEN
BACKGROUND: The endocannabinoids are emerging as natural brain protective substances that exert potentially beneficial effects in several neurological disorders by virtue of their hypothermic, immunomodulatory, vascular, antioxidant, and antiapoptotic actions. This study was undertaken to assess whether preventing the deactivation of the endocannabinoid 2-arachidonoylglycerol (2-AG) with the monoacylglycerol lipase (MAGL) inhibitor URB602 can provide neuroprotective effects in hypoxia-ischemia (HI)-induced brain injury. METHODS: URB602 was administered into the right lateral ventricle 30 min before 7-day-old pup rats were subjected to HI. The neuroprotective effect was evaluated on postnatal day (PN) 14 or at adulthood (PN80) using behavioral and histological analyses. Activated caspase-3 expression and propidium iodide labeling were assessed as indexes of apoptotic and necrotic cell death, respectively. RESULTS: Pretreatment with URB602 reduced apoptotic and necrotic cell death, as well as the infarct volume measured at PN14. At adulthood, URB602-treated HI animals performed better at the T-maze and the Morris maze, and also showed a significant reduction of brain damage. CONCLUSION: These results demonstrate that a pretreatment with URB602 significantly reduces brain damage and improves functional outcome, indicating that endocannabinoid-degrading enzymes may represent an important target for neuroprotection in neonatal ischemic brain injury.
Asunto(s)
Compuestos de Bifenilo/farmacología , Encéfalo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Monoacilglicerol Lipasas/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Ácidos Araquidónicos/metabolismo , Conducta Animal/efectos de los fármacos , Compuestos de Bifenilo/administración & dosificación , Encéfalo/enzimología , Encéfalo/patología , Encéfalo/fisiopatología , Caspasa 3/metabolismo , Modelos Animales de Enfermedad , Endocannabinoides/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/administración & dosificación , Femenino , Glicéridos/metabolismo , Hipoxia-Isquemia Encefálica/enzimología , Hipoxia-Isquemia Encefálica/patología , Hipoxia-Isquemia Encefálica/fisiopatología , Hipoxia-Isquemia Encefálica/psicología , Inyecciones Intraventriculares , Monoacilglicerol Lipasas/metabolismo , Necrosis , Fármacos Neuroprotectores/administración & dosificación , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
The endocannabinoid (EC) system is a complex cell-signaling system that participates in a vast number of biological processes since the prenatal period, including the development of the nervous system, brain plasticity, and circuit repair. This neuromodulatory system is also involved in the response to endogenous and environmental insults, being of special relevance in the prevention and/or treatment of vascular disorders, such as stroke and neuroprotection after neonatal brain injury. Perinatal hypoxia-ischemia leading to neonatal encephalopathy is a devastating condition with no therapeutic approach apart from moderate hypothermia, which is effective only in some cases. This overview, therefore, gives a current description of the main components of the EC system (including cannabinoid receptors, ligands, and related enzymes), to later analyze the EC system as a target for neonatal neuroprotection with a special focus on its neurogenic potential after hypoxic-ischemic brain injury.
RESUMEN
Dysfunctional autophagy is linked to neuronal damage in ischemia/reperfusion injury. The Ras-related protein 7 (Rab7), a member of the Rab family of small GTPases, appears crucial for the progression of the autophagic flux, and its activity is strictly interconnected with the histone deacetylase Silent information regulator 1 (Sirt1) and transcription factor Forkhead box class O1 (FoxO1). The present study assessed the neuroprotective role of melatonin in the modulation of the Sirt1/FoxO1/Rab7 axis in HT22 cells and organotypic hippocampal cultures exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin re-established physiological levels of autophagy and reduced propidium iodide-positive cells, speeding up autophagosome (AP) maturation and increasing lysosomal activity. Our study revealed that melatonin modulates autophagic pathways, increasing the expression of both Rab7 and FoxO1 and restoring the Sirt1 expression affected by OGD/R. In addition, the Sirt1 inhibitor EX-527 significantly reduced Rab7, Sirt1, and FoxO1 expression, as well as autolysosomes formation, and blocked the neuroprotective effect of melatonin. Overall, our findings provide, for the first time, new insights into the neuroprotective role of melatonin against ischemic injury through the activation of the Sirt1/FoxO1/Rab7 axis.
Asunto(s)
Melatonina , Humanos , Melatonina/farmacología , Sirtuina 1/metabolismo , Autofagosomas/metabolismo , Isquemia , Hipocampo/metabolismo , Proteína Forkhead Box O1/metabolismoRESUMEN
The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in cell suspensions from several rat tissues obtained by two different methods, automated-mechanical and enzymatic disaggregation. Flow cytometric, confocal, and ultrastructural (TEM) analyses were applied to the spleen, testis, liver and other tissues. Samples were treated by an enzymatic trypsin solution or processed by the Medimachine II (MMII). The automated-mechanical and enzymatic disaggregation procedures have shown to work similarly in some tissues, which displayed comparable amounts of apoptotic/necrotic cells. However, cells obtained by the enzyme-free Medimachine II protocols show a better preservation lysosome and mitochondria labeling, whereas the enzymatic gentle dissociation appears to constantly induce a lower amount of intracellular ROS; nevertheless, lightly increased ROS can be recognized as a complimentary signal to promote cell survival. Therefore, MMII represents a simple, fast, and standardized method for tissue processing, which allows to minimize bias arising from the operator's ability. Our study points out technical issues to be adopted for specific organs and tissues to obtain functional cells.
Asunto(s)
Testículo , Animales , Recuento de Células , Supervivencia Celular , Citometría de Flujo/métodos , Masculino , Ratas , Especies Reactivas de OxígenoRESUMEN
Previous studies have shown that simvastatin (Sim) has neuroprotective effects in a neonatal model of hypoxia-ischemia (HI)-induced brain injury when administered before but not after HI, pointing to the preconditioning (PC)-like effects of the statin. The present study aimed to gain more insight into the PC-like effect of Sim by studying the role of autophagy and its modulation by mTOR and SIRT1 in neuroprotection. Sim potentiated the autophagy response induced by neonatal HI, as shown by the increased expression of both microtubule-associated protein 1 light chain 3 (LC3) and beclin 1, increased monodansylcadaverine (MDC) labeling, and reduced expression of p62. The autophagy inhibitor 3-methyladenine (3MA) completely blocked the neuroprotective effect of Sim. Two hours after HI, there was a reduction in the activity of mTORC1 and a concomitant increase in that of mTORC2. Sim preconditioning further decreased the activity of mTORC1, but did not affect that of mTORC2. However, 24 h after injury, mTORC2 activity was significantly preserved in Sim-treated rats. Sim preconditioning also prevented the depletion of SIRT1 induced by HI, an effect that was completely blocked by 3MA. These data show that Sim preconditioning may modulate autophagy and survival pathways by affecting mTORC1, mTORC2, and SIRT1 activities. This study provides further preclinical evidence of the PC-like effect of statins in brain tissue, supporting their beneficial effects in improving stroke outcome after prophylactic treatments.
Asunto(s)
Autofagia/efectos de los fármacos , Daño Encefálico Crónico/prevención & control , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Simvastatina/farmacología , Sirtuina 1/efectos de los fármacos , Sirtuina 1/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Animales , Animales Recién Nacidos , Daño Encefálico Crónico/etiología , Daño Encefálico Crónico/patología , Hipoxia-Isquemia Encefálica/complicaciones , Hipoxia-Isquemia Encefálica/patología , Precondicionamiento Isquémico , Diana Mecanicista del Complejo 1 de la Rapamicina/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/efectos de los fármacos , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Ratas , Ratas Sprague-Dawley , Simvastatina/antagonistas & inhibidoresRESUMEN
The number of functions controlled by the endocannabinoid system in health and disease continues growing over the years. In the brain, these include the modulation of harmful events such as glutamate excitotoxicity, oxidative stress, and inflammation, mainly regulated by activation/blockade of CB1/CB2 cannabinoid receptors. In the present work, we evaluated the capacity of the CB1 antagonist/CB2 agonist synthetic cannabinoid URB447 on reducing neurodegeneration after brain injury. By using a model of hypoxia-ischemia (HI) in neonatal rats, we found that URB447 strongly reduced brain injury when administered before HI. A comparable effect was observed with the CB1 antagonist SR141716A, whereas the CB1 agonist WIN-55,212-2 reduced the effect of URB447. When administered 3 h after HI, which is considered a clinically feasible therapeutic window to treat perinatal brain injury in humans, URB447 reduced neurodegeneration and white matter damage. Markers of astrogliosis and microglial activation also appeared reduced. These results confirm the important role played by the endocannabinoid system in the neurodegenerative process and strongly encourage further research into the mechanisms of URB447-induced neuroprotection.