RESUMEN
Considerable efforts are currently made to develop strategies that boost endogenous recovery once a stroke has occurred. Owing to their restorative properties, neurotrophic factors are attractive candidates that capitalize on endogenous response mechanisms. Non-conventional growth factors cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) promote neuronal survival and reduce neurological deficits in the acute phase of ischemic stroke in mice. Their effects on endogenous repair and recovery mechanisms in the stroke recovery phase were so far unknown. By intracerebroventricular delivery of CDNF or MANF starting 3 days post-stroke (1 µg/day for 28 days via miniosmotic pumps), we show that delayed CDNF and MANF administration promoted functional neurological recovery assessed by a battery of behavioral tests, increased long-term neuronal survival, reduced delayed brain atrophy, glial scar formation, and, in case of CDNF but not MANF, increased endogenous neurogenesis in the perilesional brain tissue. Besides, CDNF and MANF administration increased long-distance outgrowth of terminal axons emanating from the contralesional pyramidal tract, which crossed the midline to innervate ipsilesional facial nucleus. This plasticity promoting effect was accompanied by downregulation of the axonal growth inhibitor versican and the guidance molecules ephrin B1 and B2 in the previously ischemic hemisphere at 14 dpi, which represents a sensitive time-point for axonal growth. CDNF and MANF reduced the expression of the proinflammatory cytokines IL1ß and TNFα in both hemispheres. The effects of non-conventional growth factors in the ischemic brain should further be examined since they might help to identify targets for restorative stroke therapy.
Asunto(s)
Dopamina , Accidente Cerebrovascular , Animales , Ratones , Astrocitos/metabolismo , Axones , Encéfalo/metabolismo , Dopamina/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacologíaRESUMEN
The phosphodiesterase (PDE) superfamily comprises enzymes responsible for the cAMP and cGMP degradation to AMP and GMP. PDEs are abundant in the brain, where they are involved in several neuronal functions. High PDE10A abundance was previously observed in the striatum; however its consequences for stroke recovery were unknown. Herein, we evaluated the effects of PDE10A deactivation by TAK-063 (0.3 or 3 mg/kg, initiated 72 h post-stroke) in mice exposed to intraluminal middle cerebral artery occlusion. We found that PDE10A deactivation over up to eight weeks dose-dependently increased long-term neuronal survival, angiogenesis, and neurogenesis in the peri-infarct striatum, which represents the core of the middle cerebral artery territory, and reduced astroglial scar formation, whole brain atrophy and, more specifically, striatal atrophy. Functional motor-coordination recovery and the long-distance plasticity of pyramidal tract axons, which originate from the contralesional motor cortex and descend through the contralesional striatum to innervate the ipsilesional facial nucleus, were enhanced by PDE10A deactivation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed a set of dopamine receptor-related and neuronal plasticity-related PDE10A targets, which were elevated (e.g., protein phosphatase-1 regulatory subunit 1B) or reduced (e.g., serine/threonine protein phosphatase 1α, ß-synuclein, proteasome subunit α2) by PDE10A deactivation. Our results identify PDE10A as a therapeutic target that critically controls post-ischemic brain tissue remodeling and plasticity.
Asunto(s)
Ataque Isquémico Transitorio , Hidrolasas Diéster Fosfóricas , Accidente Cerebrovascular , Adenosina Monofosfato/metabolismo , Animales , Atrofia , Cromatografía Liquida , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Ratones , Hidrolasas Diéster Fosfóricas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Fosfatasa 1/metabolismo , Tractos Piramidales/metabolismo , Receptores Dopaminérgicos/metabolismo , Accidente Cerebrovascular/tratamiento farmacológico , Espectrometría de Masas en Tándem , Sinucleína beta/metabolismoRESUMEN
Lithium, in addition to its effect on acute and long-term bipolar disorder, is involved in neuroprotection after ischemic stroke. Yet, its mechanism of action is still poorly understood, which was only limited to its modulatory effect on GSK pathway. Therefore, we initially analyzed the dose-dependent effects of lithium on neurological deficits, infarct volume, brain edema and blood-brain barrier integrity, along with neuronal injury and survival in mice subjected to focal cerebral ischemia. Thereafter, we investigated the involvement of the PI3K/Akt and MEK signal transduction pathways and their components. Our observations revealed that 2 mmol/kg lithium significantly improved post-ischemic brain tissue survival. Although, 2 mmol/kg lithium had no negative effect on brain microcirculation, 5 and 20 mmol/kg lithium reduced brain perfusion. Furthermore, supratherapeutic dose of lithium in 20 mmol/kg lead to animal death. In addition, improvement of brain perfusion with L-arginine, did not change the effect of 5 mmol/kg lithium on brain injury. Additionally, post-stroke blood-brain barrier leakage, hemodynamic impairment and apoptosis have been reversed by lithium treatment. Interestingly, lithium-induced neuroprotection was associated with increased phosphorylation of Akt at Thr308 and suppressed GSK-3ß phosphorylation at Ser9 residue. Lithium upregulated Erk-2 and downregulated JNK-2 phosphorylation. To distinguish whether neuroprotective effects of lithium are modulated by PI3K/Akt or MEK, we sequentially blocked these pathways and demonstrated that the neuroprotective activity of lithium persisted during MEK/ERK inhibition, whereas PI3K/Akt inhibition abolished neuroprotection. Collectively, we demonstrated lithium exerts its post-stroke neuroprotective activity via the PI3K/Akt pathway, specifically via Akt phosphorylation at Thr308, but not via MEK/ERK.
Asunto(s)
Isquemia Encefálica , Fármacos Neuroprotectores , Accidente Cerebrovascular , Animales , Apoptosis , Isquemia Encefálica/metabolismo , Infarto Cerebral , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Litio/farmacología , Litio/uso terapéutico , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neuroprotección , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Accidente Cerebrovascular/complicacionesRESUMEN
Phosphodiesterase 10A (PDE10A) hydrolyzes adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). It is highly expressed in the striatum. Recent evidence implied that PDE10A may be involved in the inflammatory processes following injury, such as ischemic stroke. Its role in ischemic injury was unknown. Herein, we exposed mice to 90 or 30-min middle cerebral artery occlusion, followed by the delivery of the highly selective PDE10A inhibitor TAK-063 (0.3 mg/kg or 3 mg/kg) immediately after reperfusion. Animals were sacrificed after 24 or 72 h, respectively. Both TAK-063 doses enhanced neurological function, reduced infarct volume, increased neuronal survival, reduced brain edema, and increased blood-brain barrier integrity, alongside cerebral microcirculation improvements. Post-ischemic neuroprotection was associated with increased phosphorylation (i.e., activation) of pro-survival Akt, Erk-1/2, GSK-3α/ß and anti-apoptotic Bcl-xL abundance, decreased phosphorylation of pro-survival mTOR, and HIF-1α, MMP-9 and pro-apoptotic Bax abundance. Interestingly, PDE10A inhibition reduced inflammatory cytokines/chemokines, including IFN-γ and TNF-α, analyzed by planar surface immunoassay. In addition, liquid chromatography-tandem mass spectrometry revealed 40 proteins were significantly altered by TAK-063. Our study established PDE10A as a target for ischemic stroke therapy.
Asunto(s)
Edema Encefálico/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacos , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Inhibidores de Fosfodiesterasa/uso terapéutico , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Edema Encefálico/metabolismo , Modelos Animales de Enfermedad , Accidente Cerebrovascular Isquémico/metabolismo , Ratones , Microcirculación/efectos de los fármacos , Neuroprotección/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazoles/farmacología , Pirazoles/uso terapéutico , Piridazinas/farmacología , Piridazinas/uso terapéutico , Transducción de Señal/efectos de los fármacosRESUMEN
Apart from its metabolic or physiological functions, melatonin has a potent cytoprotective activity in the physiological and pathological conditions. It is synthetized by the pineal gland and released into the blood circulation but particularly cerebrospinal fluid in a circadian manner. It can also easily diffuse through cellular membranes due its small size and lipophilic structure. Its cytoprotective activity has been linked to its potent free radical scavenger activity with the desirable characteristics of a clinically- reliable antioxidant. Melatonin detoxifies oxygen and nitrogen-based free radicals and oxidizing agents, including the highly toxic hydroxyl-and peroxynitrite radicals, initiating cellular damage. However, the cytoprotective activity of melatonin is complex and cannot be solely limited to its free radical scavenger activity. It regulates cellular signaling pathways through receptor dependent and independent mechanisms. Most of these downstream molecules, such as PI3K/AKT pathway components, also contribute to the cytoprotective effects of melatonin. In this term, melatonin is a promising molecule for the treatment of neurodegenerative disorders, such as ischemic stroke, which melatonin reduces ischemic brain injury in animal models of ischemic stroke. It regulates also circadian rhythm proteins after ischemic stroke, playing roles in cellular survival. In this context, present article summarizes the possible role of melatonin in the pathophysiological events after ischemic stroke.
Asunto(s)
Apoptosis/efectos de los fármacos , Accidente Cerebrovascular Isquémico , Melatonina , Animales , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Radicales Libres/metabolismo , Humanos , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/fisiopatología , Melatonina/metabolismo , Melatonina/farmacología , Melatonina/fisiología , Ratones , Sustancias Protectoras/metabolismo , Sustancias Protectoras/farmacología , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Axonal regeneration of the inferior alveolar nerve (IAN) is a therapeutic target for functional recovery after peripheral nerve injury. Rifampicin exerts anti-apoptotic, anti-inflammatory, and anti-oxidant effects on nerve tissues that may enhance functional recovery after peripheral nerve injury. The aim of the present study was to evaluate the therapeutic effects of systemic rifampicin following IAN crush injury. Following the nerve crush injuries of the IAN, 24 Sprague-Dawley rats were randomly divided into three groups to receive daily intraperitoneal injections of either vehicle, 5 mg kg-1 rifampicin, or 20 mg kg-1 rifampicin. Twenty-four days after induction of nerve injuries, Fluorogold (FG) was injected over the mental foramen for the evaluation of neuronal survival. At the end of the four-week period, histologic and histomorphometric examination of IAN samples were performed and FG positive cells were counted in the trigeminal ganglion sections. FG positive cells were significantly more frequent in the 20 and 5 mg kg-1 rifampicin groups than in the vehicle-treated group. Electron microscopic analyses revealed that the percentage of axons with optimum g-ratio was significantly lower in the vehicle group than in both treatment groups. In conclusion, systemic rifampicin treatment can enhance peripheral nerve regeneration.
Asunto(s)
Lesiones por Aplastamiento , Nervio Mandibular , Animales , Lesiones por Aplastamiento/tratamiento farmacológico , Regeneración Nerviosa , Ratas , Ratas Sprague-Dawley , Rifampin , Ganglio del TrigéminoRESUMEN
BACKGROUND: Newborn hypoxia ischemia (HI) is one of the most prevalent cases in the emergency and can result from fetal hypoxia during delivery. In HI, restricted blood supply to the fetal brain may cause epilepsy or mental disorders. METHODS: In the present study, seven-day-old pups were subjected HI and treated with different normobaric oxygen (NBO) concentrations (21%, 70% or 100%). In the acute phase, we analyzed infarct area, disseminate neuronal injury and surviving neurons. In addition, we studied the regulation of PTEN and MMP-9 proteins which were suggested to be activated by HI in the ischemic tissue. Moreover, long-term effects of NBO treatments were evaluated with open field, rotarod and Barnes maze tests. We also examined axonal plasticity with EGFP-AAV injection. RESULTS: Here, we demonstrate that hyperoxic NBO concentration causes an increase in cellular survival and a decrease in the number of apoptotic cells, meanwhile inhibiting the proteins involved in cellular death mechanisms. Moreover, we found that hyperoxia decreases anxiety, promotes motor coordination and improve spatial learning and memory. Notably that axonal sprouting was promoted by hyperoxia. CONCLUSION: Our data suggest that NBO is a promising approach for the treatment of newborn HI, which encourage proof-of-concept studies in newborn.
Asunto(s)
Hipoxia-Isquemia Encefálica/terapia , Enfermedades del Recién Nacido/terapia , Actividad Motora/fisiología , Plasticidad Neuronal/fisiología , Terapia por Inhalación de Oxígeno , Recuperación de la Función/fisiología , Aprendizaje Espacial/fisiología , Animales , Animales Recién Nacidos , Conducta Animal/fisiología , Supervivencia Celular/fisiología , Modelos Animales de Enfermedad , Humanos , Hipoxia-Isquemia Encefálica/metabolismo , Recién Nacido , Metaloproteinasa 9 de la Matriz/metabolismo , Neuronas/fisiología , Terapia por Inhalación de Oxígeno/métodos , Fosfohidrolasa PTEN/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The circadian rhythm is driven by a master clock within the suprachiasmatic nucleus which regulates the rhythmic secretion of melatonin. Bmal1 coordinates the rhythmic expression of transcriptome and regulates biological activities, involved in cell metabolism and aging. However, the role of Bmal1 in cellular- survival, signaling, its interaction with intracellular proteins, and how melatonin regulates its expression is largely unclear. Here we observed that melatonin increases the expression of Bmal1 and both melatonin and Bmal1 increase cellular survival after oxygen glucose deprivation (OGD) while the inhibition of Bmal1 resulted in the decreased cellular survival without affecting neuroprotective effects of melatonin. By using a planar surface immunoassay for PI3K/AKT signaling pathway components, we revealed that both melatonin and Bmal1 increased phosphorylation of AKT, ERK-1/2, PDK1, mTOR, PTEN, GSK-3αß, and p70S6K. In contrast, inhibition of Bmal1 resulted in decreased phosphorylation of these proteins, which the effect of melatonin on these signaling molecules was not affected by the absence of Bmal1. Besides, the inhibition of PI3K/AKT decreased Bmal1 expression and the effect of melatonin on Bmal1 after both OGD in vitro and focal cerebral ischemia in vivo. Our data demonstrate that melatonin controls the expression of Bmal1 via PI3K/AKT signaling, and Bmal1 plays critical roles in cellular survival via activation of survival kinases.
Asunto(s)
Factores de Transcripción ARNTL/metabolismo , Melatonina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Glucosa/deficiencia , Masculino , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Oxígeno , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Indolamine melatonin structurally resembles non-covalent proteasome inhibitors; however, the role of ubiquitin proteasome system (UPS) in neuronal survival and how melatonin carries out UPS inhibition remain largely unknown. With the use of melatonin treated cells, we evaluated the expression of Nedd4-1, an E3 ligase, how melatonin regulates its activity and its relationship with neuronal survival. Nedd4-1 was upregulated in the hypoxic condition in both control and Nedd4-1 overexpressed cells and melatonin treatment reversed its expression in both normoxic and hypoxic conditions, which was associated with increased cellular survival. Melatonin had no effect on the expression of Nedd4-1 at mRNA level. However, when melatonin was administered along with protein synthesis inhibitor cycloheximide, protein level of Nedd4-1 was further reduced, indicating that melatonin possibly downregulates Nedd4-1 after its synthesis. Notably, co-immunoprecipitation analyses followed by Liquid chromatography-Mass Spectrometry (LC-MS/MS) revealed that melatonin may dissociate ribosomal proteins, such as RS19, RL23A, and nucleophosmin from Nedd4-1, while 40S ribosomal protein S7 and 60S ribosomal protein L35 came into contact with Nedd4-1 upon melatonin treatment. By using IPA analyses, we obtained further data indicated novel target molecules of melatonin in hypoxic conditions, including OTOF, SF3B2, IPO5, ST13, FGFR3, Mx1/Mx2, playing roles in RNA splicing and trafficking, growth factor and interferon signaling. Here, we described a new insight into the role of melatonin in UPS functioning by proposing a molecular mechanism through which melatonin regulates Nedd4-1.
Asunto(s)
Supervivencia Celular , Melatonina/fisiología , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Animales , Western Blotting , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Regulación hacia Abajo , Cromatografía de Gases y Espectrometría de Masas , Hipoxia/metabolismo , Inmunoprecipitación , Melatonina/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/fisiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Repetitive transcranial magnetic stimulation (rTMS) has gained interest as a non-invasive treatment for stroke based on the data promoting its effects on functional recovery. However, the exact action mechanisms by which the rTMS exert beneficial effects in cellular and molecular aspect are largely unknown. To elucidate the effects of high- and low-frequency rTMS in the acute-ischemic brain, we examined how rTMS influences injury development, cerebral blood flow (CBF), DNA fragmentation, neuronal survival, pro- and anti-apoptotic protein activations after 30 and 90 min of focal cerebral ischemia. In addition, inflammation, angiogenesis, growth factors and axonal outgrowth related gene expressions, were analyzed. Furthermore, we have investigated the effects of rTMS on post-acute ischemic brain, particularly on spontaneous locomotor activity, perilesional tissue remodeling, axonal sprouting of corticobulbar tracts, glial scar formation and cell proliferation, in which rTMS was applied starting 3 days after the stroke onset for 28 days. In the high-frequency rTMS received animals reduced DNA fragmentation, infarct volume and improved CBF were observed, which were associated with increased Bcl-xL activity and reduced Bax, caspase-1, and caspase-3 activations. Moreover, increased angiogenesis, growth factors; and reduced inflammation and axonal sprouting related gene expressions were observed. These results correlated with reduced microglial activation, neuronal degeneration, glial scar formation and improved functional recovery, tissue remodeling, contralesional pyramidal tract plasticity and neurogenesis in the subacute rTMS treated animals. Overall, we propose that high-frequency rTMS in stroke patients can be used to promote functional recovery by inducing the endogenous repair and recovery mechanisms of the brain.
RESUMEN
Traumatic brain injury (TBI) is the leading cause of mortality and morbidity in young adults and children in the industrialized countries; however, there are presently no FDA approved therapies. TBI results in oxidative stress due to the overproduction of reactive oxygen species and overwhelming of the endogenous antioxidant mechanisms. Recently, it has been reported that antioxidants including phytochemicals have a protective role against oxidative damage and inflammation after TBI. To analyze the effects of a naturally occurring antioxidant molecule, allyl isothiocyanate (AITC), on the nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor kappa B (NF-κB) signaling pathways in TBI, a cryogenic injury model was induced in mice. Here, we showed that AITC administered immediately after the injury significantly decreased infarct volume and blood-brain barrier (BBB) permeability. Protein levels of proinflammatory cytokines interleukin-1ß (IL1ß) and interleukin-6 (IL6), glial fibrillary acidic protein (GFAP) and NF-κB were decreased, while Nrf2, growth-associated protein 43 (GAP43) and neural cell adhesion molecule levels were increased with AITC when compared with vehicle control. Our results demonstrated that the antioxidant molecule AITC, when applied immediately after TBI, provided beneficial effects on inflammatory processes while improving infarct volume and BBB permeability. Increased levels of plasticity markers, as well as an antioxidant gene regulator, Nrf2, by AITC, suggest that future studies are warranted to assess the protective activities of dietary or medicinal AITC in clinical studies.
Asunto(s)
Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Isotiocianatos/farmacología , Animales , Antioxidantes/farmacología , Lesiones Encefálicas/tratamiento farmacológico , Citocinas/metabolismo , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Hemo-Oxigenasa 1/efectos de los fármacos , Inflamación/tratamiento farmacológico , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-1beta/efectos de los fármacos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Isotiocianatos/metabolismo , Masculino , Proteínas de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/efectos de los fármacos , FN-kappa B/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Occurrence of stroke cases displays a time-of-day variation in human. However, the mechanism linking circadian rhythm to the internal response mechanisms against pathophysiological events after ischemic stroke remained largely unknown. To this end, temporal changes in the susceptibility to ischemia/reperfusion (I/R) injury were investigated in mice in which the ischemic stroke induced at four different Zeitgeber time points with 6-h intervals (ZT0, ZT6, ZT12, and ZT18). Besides infarct volume and brain swelling, neuronal survival, apoptosis, ischemia, and circadian rhythm related proteins were examined using immunohistochemistry, Western blot, planar surface immune assay, and liquid chromatography-mass spectrometry tools. Here, we present evidence that midnight (ZT18; 24:00) I/R injury in mice resulted in significantly improved infarct volume, brain swelling, neurological deficit score, neuronal survival, and decreased apoptotic cell death compared with ischemia induced at other time points, which were associated with increased expressions of circadian proteins Bmal1, PerI, and Clock proteins and survival kinases AKT and Erk-1/2. Moreover, ribosomal protein S6, mTOR, and Bad were also significantly increased, while the levels of PRAS40, negative regulator of AKT and mTOR, and phosphorylated p53 were decreased at this time point compared to ZT0 (06:00). Furthermore, detailed proteomic analysis revealed significantly decreased CSKP, HBB-1/2, and HBA levels, while increased GNAZ, NEGR1, IMPCT, and PDE1B at midnight as compared with early morning. Our results indicate that nighttime I/R injury results in less severe neuronal damage, with increased neuronal survival, increased levels of survival kinases and circadian clock proteins, and also alters the circadian-related proteins.
Asunto(s)
Factores de Transcripción ARNTL/biosíntesis , Isquemia Encefálica/metabolismo , Ritmo Circadiano/fisiología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Accidente Cerebrovascular/metabolismo , Animales , Isquemia Encefálica/patología , Supervivencia Celular/fisiología , Relojes Circadianos/fisiología , Fragmentación del ADN , Masculino , Ratones , Ratones Endogámicos BALB C , Neuronas/patología , Accidente Cerebrovascular/patologíaRESUMEN
Conflicting data in the literature about the function of P2X7R in survival following ischemia necessitates the conductance of in-depth studies. To investigate the impacts of activation vs inhibition of the receptor on neuronal survival as well as the downstream signaling cascades, in addition to optic nerve transection (ONT), 30min and 90min of middle cerebral artery occlusion (MCAo) models were performed in mice. Intracellular calcium levels were assessed in primary cortical neuron cultures. Here, we show that P2X7R antagonist Brilliant Blue G (BBG) decreased DNA fragmentation, infarct volume, brain swelling, neurological deficit scores and activation of microglial cells after focal cerebral ischemia. BBG also significantly increased the number of surviving retinal ganglion cells (RGCs) after ONT and the number of surviving neurons following MCAo. Importantly, receptor agonist BzATP resulted in increased activation of microglial cells and induced phosphorylation of ERK, AKT and JNK. These results indicated that inhibition of P2X7R with BBG promoted neuronal survival, not through the activation of survival kinase pathways, but possibly by improved intracellular Ca2+ overload and decreased the levels of Caspase 1, IL-1ß and Bax proteins. On the other hand, BzATP-mediated increased number of activated microglia and increased survival kinase levels in addition to increased caspase-1 and IL-1ß levels indicate the complex nature of the P2X7 receptor-mediated signaling in neuronal injury.
Asunto(s)
Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Neuronas/patología , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/patología , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/uso terapéutico , Animales , Animales Recién Nacidos , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Edema Encefálico/etiología , Isquemia Encefálica/tratamiento farmacológico , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Citocinas/metabolismo , Fragmentación del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Infusiones Intraventriculares , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , Neuronas/efectos de los fármacos , Traumatismos del Nervio Óptico/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Colorantes de Rosanilina/metabolismoRESUMEN
Apart from its potent antioxidant property, recent studies have revealed that melatonin promotes PI3K/Akt phosphorylation following focal cerebral ischemia (FCI) in mice. However, it is not clear (i) whether increased PI3K/Akt phosphorylation is a concomitant event or it directly contributes to melatonin's neuroprotective effect, and (ii) how melatonin regulates PI3K/Akt signaling pathway after FCI. In this study, we showed that Akt was intensively phosphorylated at the Thr308 activation loop as compared with Ser473 by melatonin after FCI. Melatonin treatment reduced infarct volume, which was reversed by PI3K/Akt inhibition. However, PI3K/Akt inhibition did not inhibit melatonin's positive effect on brain swelling and IgG extravasation. Additionally, phosphorylation of mTOR, PTEN, AMPKα, PDK1 and RSK1 were increased, while phosphorylation of 4E-BP1, GSK-3α/ß, S6 ribosomal protein were decreased in melatonin treated animals. In addition, melatonin decreased apoptosis through reduced p53 phosphorylation by the PI3K/Akt pathway. In conclusion, we demonstrated the activation profiles of PI3K/Akt signaling pathway components in the pathophysiological aspect of ischemic stroke and melatonin's neuroprotective activity. Our data suggest that Akt phosphorylation, preferably at the Thr308 site of the activation loop via PDK1 and PTEN, mediates melatonin's neuroprotective activity and increased Akt phosphorylation leads to reduced apoptosis.
Asunto(s)
Antioxidantes/administración & dosificación , Isquemia Encefálica/tratamiento farmacológico , Melatonina/administración & dosificación , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Antioxidantes/farmacología , Isquemia Encefálica/inmunología , Isquemia Encefálica/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Inmunoglobulina G/metabolismo , Melatonina/farmacología , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-akt/química , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Transducción de Señal/efectos de los fármacos , Treonina/metabolismoRESUMEN
OBJECTIVES: Folic acid (FA) is physiologically important in mammals and is a common vitamin supplement used during pregnancy and lactation. Numerous studies have reported that FA significantly improves endothelial function. The blood-brain barrier (BBB) plays an important role in maintaining the microenvironment required for neuronal function, but its unique structure is damaged by epileptic seizures. The aim of this study was to evaluate the potential protective role of FA on BBB leakage, as well as on the reactive astrogliosis in pregnant rats and their prepubertal offspring during pentylenetetrazole (PTZ)-induced epileptic seizure. METHODS: Pregnant rats were treated with FA (5 mg/kg) and PTZ on gestational days 0-19 and 19, respectively. The pups were treated with PTZ at puberty. Evans blue was used to evaluate BBB integrity. Reactive astrogliosis was defined using immunohistochemical analysis for glial fibrillary acidic protein (GFAP). Mean arterial blood pressure (MABP) was measured at the femoral artery. RESULTS: A moderate decrease in BBB leakage was observed in FA-treated pregnant and prepubertal animals (P < 0.05). MABP was decreased significantly in pregnant rats (P < 0.05). The epilepsy-induced increase in MABP was less prominent in pregnant animals (P < 0.05). GFAP intensity decreased in PTZ-treated pregnant animals (P < 0.01) and FA-treated prepubertal rats. DISCUSSION: Our findings suggest that FA, which is used as a maternal vitamin to promote normal fetus development, may be beneficial against seizure-induced neuronal damage by decreasing BBB leakage and reactive astrogliosis in pregnant and prepubertal rats.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Ácido Fólico/farmacología , Pentilenotetrazol/efectos adversos , Convulsiones/tratamiento farmacológico , Animales , Presión Sanguínea/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Epilepsia/inducido químicamente , Epilepsia/tratamiento farmacológico , Femenino , Ácido Fólico/administración & dosificación , Proteína Ácida Fibrilar de la Glía/metabolismo , Fenómenos Fisiologicos Nutricionales Maternos , Embarazo , Ratas , Ratas Wistar , Convulsiones/inducido químicamenteRESUMEN
The tissue damage that emerges during traumatic brain injury (TBI) is a consequence of a variety of pathophysiological events, including free radical generation and over-activation of N-methyl-d-aspartate-type glutamate receptors (NMDAR). Considering the complex pathophysiology of TBI, we hypothesized that combination of neuroprotective compounds, targeting different events which appear during injury, may be a more promising approach for patients. In this context, both NMDAR antagonist memantine and free radical scavenger melatonin are safe in humans and promising agents for the treatment of TBI. Herein, we examined the effects of melatonin administered alone or in combination with memantine on the activation of signaling pathways, injury development and DNA fragmentation. Both compounds reduced brain injury moderately and the density of DNA fragmentation significantly. Notably, melatonin/memantine combination decreased brain injury and DNA fragmentation significantly, which was associated with reduced p38 and ERK-1/2 phosphorylation. As compared with melatonin and memantine groups, SAPK/JNK-1/2 phosphorylation was also reduced in melatonin/memantine combined animals. In addition, melatonin, memantine and their combination decreased iNOS activity significantly. Here, we provide evidence that melatonin/memantine combination protects brain from traumatic injury, which was associated with decreased DNA fragmentation, p38 phosphorylation and iNOS activity.
Asunto(s)
Lesiones Encefálicas/tratamiento farmacológico , Depuradores de Radicales Libres/farmacología , Melatonina/farmacología , Memantina/farmacología , Fármacos Neuroprotectores/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Infarto Encefálico/patología , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Fragmentación del ADN/efectos de los fármacos , Depuradores de Radicales Libres/uso terapéutico , Masculino , Melatonina/uso terapéutico , Memantina/uso terapéutico , Ratones Endogámicos BALB C , Fármacos Neuroprotectores/uso terapéuticoRESUMEN
In order to protect the brain before an irreversible injury occurs, penumbral oxygenation is the primary goal of current acute ischemic stroke treatment. However, hyperoxia treatment remains controversial due to the risk of free radical generation and vasoconstriction. Melatonin is a highly potent free radical scavenger that protects against ischemic stroke. Considering its anti-oxidant activity, we hypothesized that melatonin may augment the survival-promoting action of normobaric oxygen (NBO) and prevent brain infarction. Herein, we exposed mice to 30 or 90 min of intraluminal middle cerebral artery occlusion (MCAo) and evaluated the effects of NBO (70% or 100% over 90 min), administered either alone or in combination with melatonin (4 mg/kg, i.p.), on disseminate neuronal injury, neurological deficits, infarct volume, blood-brain barrier (BBB) permeability, cerebral blood flow (CBF) and cell signaling. Both NBO and particularly melatonin alone reduced neuronal injury, neurological deficits, infarct volume and BBB permeability, and increased post-ischemic CBF, evaluated by laser speckle imaging (LSI). They also improved CBF significantly in the ischemic- core and penumbra, which was associated with reduced IgG extravasation, DNA fragmentation, infarct volume, brain swelling and neurological scores. Levels of phosphorylated Akt, anti-apoptotic Bcl-xL, pro-apoptotic Bax and endothelial nitric oxide synthase (NOS) were re-regulated after combined oxygen and melatonin delivery, whereas neuronal and inducible NOS, which were increased by oxygen treatment, were not influenced by melatonin. Our present data suggest that melatonin and NBO are promising approaches for the treatment of acute-ischemic stroke, which encourage proof-of-concept studies in human stroke patients.
Asunto(s)
Antioxidantes/uso terapéutico , Isquemia Encefálica/terapia , Encéfalo/irrigación sanguínea , Depuradores de Radicales Libres/uso terapéutico , Melatonina/uso terapéutico , Oxígeno/uso terapéutico , Accidente Cerebrovascular/terapia , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiopatología , Encéfalo/patología , Edema Encefálico/patología , Fragmentación del ADN , Modelos Animales de Enfermedad , Inmunoglobulina G/sangre , Infarto de la Arteria Cerebral Media/fisiopatología , Ratones , Ratones Endogámicos BALB C , Microcirculación , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMEN
BACKGROUND/AIMS: The aim of the present study was to investigate the effect of combination of aliskiren with paricalcitol on experimental diabetic nephropathy (DN) model in rats. METHODS: Forty male Sprague Dawley rats were divided into 5 groups of 8 rats each, namely the control (Group C), diabetes (Group D), aliskiren (Group A), paricalcitol (Group P), and aliskiren plus paricalcitol (Group A+P) groups. Aliskiren was given by oral-gavage at a dose of 50 mg/kg/day once daily for 12 weeks. Paricalcitol was given by intraperitoneally at a dose of 0,4 µg/kg/three day of week for 12 weeks. Renal function parameters, oxidative stress biomarkers, mRNA expression of renin-angiotensin system parameters and kidney histology were determined. RESULTS: Group A+P had lower mean albümin-to-creatinine ratio (ACR) (p=0.004) as well as higher creatinine clearance (CCr) (p<0.005) than the diabetic rats (Group D). Combination therapy significantly increased CCr (Group A+P vs. Group A, p<0.005; Group A+P vs. Group P, p=0.022) and reduced ACR (Group A+P vs. Group A, p=0.018; Group A+P vs. Group P, p<0.005) when compared to monotherapy. Serum malondialdehyde levels were significantly lower (p=0.004); glutathion levels (p=0.003), glutathion peroxidase (p=0.004) and superoxide dismutase (p<0.005) activities were significantly higher in group A+P than in group D. The mean scores of mRNA expression of renin (p<0.005), angiotensin II (p=0.012) and angiotensin type 1 receptor (p=0.018) in group A+P were significantly lower. Although combination therapy showed no additional effect on oxidative system, renin-angiotensin system and renal histology, aliskiren plus paricalcitol significantly decreased interstitial fibrosis volume when compared to monotherapy (Group A+P vs. Group A, p<0.005; Group A+P vs. Group P, p=0.002). CONCLUSION: Our data seem to suggest a potential role of aliskiren plus paricalcitol acting synergystically for reducing the progression of diabetic nephropathy in an experimental rat model.
Asunto(s)
Amidas/uso terapéutico , Antihipertensivos/uso terapéutico , Conservadores de la Densidad Ósea/uso terapéutico , Nefropatías Diabéticas/tratamiento farmacológico , Ergocalciferoles/uso terapéutico , Fumaratos/uso terapéutico , Animales , Antioxidantes/metabolismo , Biomarcadores/sangre , Nefropatías Diabéticas/patología , Progresión de la Enfermedad , Quimioterapia Combinada , Riñón/patología , Pruebas de Función Renal , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Sistema Renina-Angiotensina/efectos de los fármacosRESUMEN
Pea3 subfamily of E-twenty six transcription factors consist of three major -exhibit branching morphogenesis, the function of Pea3 family in nervous system development and regeneration is only beginning to unfold. In this study, we provide evidence that Pea3 can directs neurite extension and axonal outgrowth in different model systems, and that Serine 90 is important for this function. We have also identified neurofilament-L and neurofilament-M as two putative novel targets for Pea3.