RESUMO
Fingolimod, a sphingosine-1-phosphate receptor (S1PR) agonist, is clinically available to treat multiple sclerosis and is showing promise in treating stroke. We investigated if fingolimod provides long-term protection from experimental neonatal germinal matrix hemorrhage (GMH), aiming to support a potential mechanism of acute fingolimod-induced protection. GMH was induced in P7 rats by infusion of collagenase (0.3 U) into the right ganglionic eminence. Animals killed at 4 weeks post-GMH received low- or high-dose fingolimod (0.25 or 1.0 mg/kg) or vehicle, and underwent neurocognitive testing before histopathological evaluation. Subsequently, a cohort of animals killed at 72 h post-GMH received 1.0 mg/kg fingolimod; the specific S1PR1 agonist, SEW2871; or fingolimod co-administered with the S1PR1/3/4 inhibitor, VPC23019, or the Rac1 inhibitor, EHT1864. All drugs were injected intraperitoneally 1, 24, and 48 h post-surgery. At 72 h post-GMH, brain water content, extravasated Evans blue dye, and hemoglobin were measured as well as the expression levels of phospho-Akt, Akt, GTP-Rac1, Total-Rac1, ZO1, occludin, and claudin-3 determined. Fingolimod significantly improved long-term neurocognitive performance and ameliorated brain tissue loss. At 72 h post-GMH, fingolimod reduced brain water content and Evans blue dye extravasation as well as reversed GMH-induced loss of tight junctional proteins. S1PR1 agonism showed similar protection, whereas S1PR or Rac1 inhibition abolished the protective effect of fingolimod. Fingolimod treatment improved functional and morphological outcomes after GMH, in part, by tempering acute post-hemorrhagic blood-brain barrier disruption via the activation of the S1PR1/Akt/Rac1 pathway.
Assuntos
Cloridrato de Fingolimode/farmacologia , Hemorragias Intracranianas/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Água Corporal/metabolismo , Encéfalo/patologia , Química Encefálica/efeitos dos fármacos , Edema Encefálico/tratamento farmacológico , Edema Encefálico/etiologia , Cognição/efeitos dos fármacos , Feminino , Hemorragias Intracranianas/metabolismo , Hemorragias Intracranianas/psicologia , Contagem de Leucócitos , Masculino , Oxidiazóis/farmacologia , Fosfosserina/análogos & derivados , Fosfosserina/farmacologia , Gravidez , Pironas/farmacologia , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Tiofenos/farmacologia , Proteínas de Junções Íntimas/metabolismo , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
Germinal matrix hemorrhage remains the leading cause of morbidity and mortality in preterm infants in the United States with little progress made in its clinical management. Survivors are often afflicted with long-term neurological sequelae, including cerebral palsy, mental retardation, hydrocephalus, and psychiatric disorders. Blood clots disrupting normal cerebrospinal fluid circulation and absorption after germinal matrix hemorrhage are thought to be important contributors towards post-hemorrhagic hydrocephalus development. We evaluated if upregulating CD36 scavenger receptor expression in microglia and macrophages through PPARγ stimulation, which was effective in experimental adult cerebral hemorrhage models and is being evaluated clinically, will enhance hematoma resolution and ameliorate long-term brain sequelae using a neonatal rat germinal matrix hemorrhage model. PPARγ stimulation (15d-PGJ2) increased short-term PPARγ and CD36 expression levels as well as enhanced hematoma resolution, which was reversed by a PPARγ antagonist (GW9662) and CD36 siRNA. PPARγ stimulation (15d-PGJ2) also reduced long-term white matter loss and post-hemorrhagic ventricular dilation as well as improved neurofunctional outcomes, which were reversed by a PPARγ antagonist (GW9662). PPARγ-induced upregulation of CD36 in macrophages and microglia is, therefore, critical for enhancing hematoma resolution and ameliorating long-term brain sequelae.
Assuntos
Antígenos CD36/metabolismo , Hematoma/fisiopatologia , Hemorragias Intracranianas/fisiopatologia , PPAR gama/metabolismo , Anilidas/farmacologia , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/fisiopatologia , Antígenos CD36/genética , Fármacos do Sistema Nervoso Central/farmacologia , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Hematoma/tratamento farmacológico , Hematoma/patologia , Hemorragias Intracranianas/tratamento farmacológico , Hemorragias Intracranianas/patologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/fisiologia , Microglia/efeitos dos fármacos , Microglia/fisiologia , Fármacos Neuroprotetores/farmacologia , PPAR gama/antagonistas & inibidores , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , RNA Interferente Pequeno/administração & dosagem , Distribuição Aleatória , Ratos Sprague-Dawley , Regulação para CimaRESUMO
Germinal matrix hemorrhage (GMH) is the most common and devastating neurological injury of premature infants, and current treatment approaches are ineffective. Remote ischemic postconditioning (RIPC) is a method by which brief limb ischemic stimuli protect the injured brain. We hypothesized that RIPC can improve outcomes following GMH in rats. Neonatal rats (P7) were subjected to either stereotactic ganglionic eminence collagenase infusion or sham surgery. Groups were as follows: sham (n = 0), GMH non-RIPC (n = 10), GMH + 1 week RIPC (n = 10), GMH + 2 weeks RIPC (n = 10). Neurobehavior analysis at the fourth week consisted of Morris water maze (MWM) and rotarod (RR). This was followed by euthanasia for histopathology on day 28. Both 1- and 2-week RIPC showed significant improvement in FF and RR motor testing compared with untreated animals (i.e., GMH without RIPC). RIPC treatment also improved cognition (MWM) and attenuated neuropathological ventricular enlargement (hydrocephalus) in juvenile animals following GMH. RIPC is a safe and noninvasive approach that improved sensorimotor and neuropathological outcomes following GMH in rats. Further studies are needed to evaluate for mechanisms of neuroprotection.
Assuntos
Hemorragia Cerebral/terapia , Pós-Condicionamento Isquêmico/métodos , Animais , Animais Recém-Nascidos , Comportamento Animal , Encéfalo/fisiopatologia , Hemorragia Cerebral/induzido quimicamente , Hemorragia Cerebral/fisiopatologia , Modelos Animais de Doenças , Extremidades , Aprendizagem em Labirinto , Colagenase Microbiana/toxicidade , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Teste de Desempenho do Rota-RodRESUMO
Germinal matrix hemorrhage (GMH) is the most devastating neurological problem of premature infants. Current treatment strategies are ineffective and brain injury is unpreventable. Insulin-like growth factor 1 (IGF-1) is an endogenous protein shown to have multiple neuroprotective properties. We therefore hypothesized that IGF-1 would reduce brain injury after GMH. Neonatal rats (P7 age) received stereotactic collagenase into the right ganglionic eminence. The following groups were studied: (1) sham, (2) GMH + vehicle, (3) GMH + intranasal IGF-1. Three days later, the animals were evaluated using the righting-reflex (early neurobehavior), Evans blue dye leakage (blood-brain barrier (BBB) permeability), brain water content (edema), and hemoglobin assay (extent of bleeding). Three weeks later, juvenile rats were tested using a water maze (delayed neurobehavior), and then were sacrificed on day 28 for assessment of hydrocephalus (ventricular size). Intranasal IGF-1 treated animals had improved neurological function, and amelioration of BBB permeability, edema, and re-bleeding. IGF-1 may play a part in protective brain signaling following GMH, and our observed protective effect may offer new promise for treatment targeting this vulnerable patient population.
Assuntos
Comportamento Animal/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Hemorragia Cerebral/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Hemorragias Intracranianas/metabolismo , Administração Intranasal , Animais , Animais Recém-Nascidos , Barreira Hematoencefálica/metabolismo , Edema Encefálico , Hemorragia Cerebral/patologia , Hemorragia Cerebral/fisiopatologia , Modelos Animais de Doenças , Hemoglobinas/efeitos dos fármacos , Hemoglobinas/metabolismo , Hidrocefalia , Hemorragias Intracranianas/patologia , Hemorragias Intracranianas/fisiopatologia , Permeabilidade , Ratos , Ratos Sprague-DawleyRESUMO
Germinal matrix hemorrhage (GMH) is a major cause of brain damage in prematurity and has long-lasting neurological implications. The development of brain inflammation contributes to brain injury, leading to a lifetime of neurologic deficits. PAR-1 and 4 receptors are involved with inflammatory pathways after brain hemorrhage in adult models of stroke, of which cyclooxygenase-2 (COX-2) is a potential mediator. We therefore hypothesized a role for PAR-1, 4/ COX-2 signaling following GMH. Postnatal day 7 Sprague-Dawley rats were subjected to GMH induction, which entailed stereotactic collagenase infusion into the ganglionic eminence. Animals were euthanized at two time points: 72 h (short-term) or 4 weeks (long-term). Short-term COX-2 expression was evaluated in the context of PAR-1 (SCH-79797) and PAR-4 (P4pal10) inhibition. Pups in the long-term group were administered the selective COX-2 inhibitor (NS-398); and the neurobehavioral and pathological examinations were performed 4 weeks later. Pharmacological PAR-1, 4 antagonism normalized COX-2 expression following GMH and reduced hydrocephalus. Early inhibition of COX-2 by NS-398 improved long-term neurobehavioral outcomes. COX-2 signaling plays an important role in brain injury following neonatal GMH, possibly through upstream PAR-1, 4 receptor mechanisms.
Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Ciclo-Oxigenase 2/efeitos dos fármacos , Hemorragias Intracranianas/metabolismo , Oligopeptídeos/farmacologia , Pirróis/farmacologia , Quinazolinas/farmacologia , Animais , Animais Recém-Nascidos , Western Blotting , Encéfalo/patologia , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Hemorragias Intracranianas/patologia , Hemorragias Intracranianas/fisiopatologia , Ratos , Ratos Sprague-Dawley , Receptor PAR-1/antagonistas & inibidores , Receptores de Trombina/antagonistas & inibidoresRESUMO
Germinal matrix hemorrhage (GMH) is the most common cause of neurological complications of prematurity and has lasting implications. PAR-1 and PAR-4 receptors are involved with upstream signaling pathways following brain hemorrhage in adult models of stroke, of which the mammalian target of rapamycin (mTOR) is a potential downstream mediator. Therefore, we hypothesized a role for PAR-1, -4/ mTOR signaling following GMH brain injury. Postnatal day 7 Sprague-Dawley rats were subjected to GMH through stereotactic infusion of collagenase into the right ganglionic eminence. Rodents were euthanized at 72 h (short term), or 4 weeks (long term). Short-term mTOR expression was evaluated by Western blot in the context of PAR-1 (SCH-79797) and PAR-4 (P4pal10) inhibition. Pups in the long-term group were administered the selective mTOR inhibitor (rapamycin) with neurobehavioral and brain pathological examinations performed at 4 weeks. Pharmacological PAR-1, -4 antagonism normalized the increased mTOR expression following GMH. Early inhibition of mTOR by rapamycin improved long-term outcomes in rats. Mammalian-TOR signaling plays an important role in brain injury following neonatal GMH, possibly involving upstream PAR-1, -4 mechanisms.
Assuntos
Encéfalo/efeitos dos fármacos , Hemorragias Intracranianas/metabolismo , Oligopeptídeos/farmacologia , Pirróis/farmacologia , Quinazolinas/farmacologia , Receptor PAR-1/antagonistas & inibidores , Receptores de Trombina/antagonistas & inibidores , Serina-Treonina Quinases TOR/efeitos dos fármacos , Trombina/metabolismo , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Imunossupressores/farmacologia , Hemorragias Intracranianas/patologia , Hemorragias Intracranianas/fisiopatologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND AND PURPOSE: This study examines the role of thrombin's protease-activated receptor (PAR)-1, PAR-4 in mediating cyclooxygenase-2 and mammalian target of rapamycin after germinal matrix hemorrhage. METHODS: Germinal matrix hemorrhage was induced by intraparenchymal infusion of bacterial collagenase into the right ganglionic eminence of P7 rat pups. Animals were treated with PAR-1, PAR-4, cyclooxygenase-2, or mammalian target of rapamycin inhibitors by 1 hour, and ≤5 days. RESULTS: We found increased thrombin activity 6 to 24 hours after germinal matrix hemorrhage, and PAR-1, PAR-4, inhibition normalized cyclooxygenase-2, and mammalian target of rapamycin by 72 hours. Early treatment with NS398 or rapamycin substantially improved long-term outcomes in juvenile animals. CONCLUSIONS: Suppressing early PAR signal transduction, and postnatal NS398 or rapamycin treatment, may help reduce germinal matrix hemorrhage severity in susceptible preterm infants.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Lesões Encefálicas/tratamento farmacológico , Hemorragia Cerebral/tratamento farmacológico , Imunossupressores/farmacologia , Nitrobenzenos/farmacologia , Receptor PAR-1/antagonistas & inibidores , Receptores de Trombina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Sulfonamidas/farmacologia , Animais , Animais Recém-Nascidos , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/patologia , Ciclo-Oxigenase 2/metabolismo , RatosRESUMO
BACKGROUND AND PURPOSE: Blood-brain barrier disruption and consequent vasogenic edema formation codetermine the clinical course of intracerebral hemorrhage (ICH). This study examined the effect of PHA-543613, a novel α7 nicotinic acetylcholine receptor agonist, on blood-brain barrier preservation after ICH. METHODS: Male CD-1 mice, subjected to intrastriatal blood infusion, received PHA-543613 alone or in combination with α7 nicotinic acetylcholine receptor antagonist methyllycaconitine or phosphatidylinositol 3-kinase inhibitor wortmannin. RESULTS: PHA-543613 alone, but not in combination with methyllycaconitine or wortmannin, inhibited glycogen synthase kinase-3ß, thus, stabilizing ß-catenin and tight junction proteins, which was paralleled by improved blood-brain barrier stability and ameliorated neurofunctional deficits in ICH animals. CONCLUSIONS: PHA-543613 preserved blood-brain barrier integrity after ICH, possibly through phosphatidylinositol 3-kinase-Akt-induced inhibition of glycogen synthase kinase-3ß and ß-catenin stabilization.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Hemorragia Cerebral/fisiopatologia , Quinuclidinas/farmacologia , Receptores Nicotínicos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Aconitina/análogos & derivados , Aconitina/farmacologia , Androstadienos/farmacologia , Animais , Barreira Hematoencefálica/fisiologia , Hemorragia Cerebral/metabolismo , Claudina-3/metabolismo , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta , Masculino , Camundongos , Camundongos Endogâmicos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Wortmanina , beta Catenina/metabolismoRESUMO
BACKGROUND AND PURPOSE: This study investigated whether isoflurane ameliorates neurological sequelae after germinal matrix hemorrhage (GMH) through activation of the cytoprotective sphingosine kinase/sphingosine-1-phosphate receptor/Akt pathway. METHODS: GMH was induced in P7 rat pups by intraparenchymal infusion of bacterial collagenase (0.3 U) into the right hemispheric germinal matrix. GMH animals received 2% isoflurane either once 1 hour after surgery or every 12 hours for 3 days. Isoflurane treatment was then combined with sphingosine-1-phosphate receptor-1/2 antagonist VPC23019 or sphingosine kinase 1/2 antagonist N,N-dimethylsphingosine. RESULTS: Brain protein expression of sphingosine kinase-1 and phosphorylated Akt were significantly increased after isoflurane post-treatment, and cleaved caspase-3 was decreased at 24 hours after surgery, which was reversed by the antagonists. Isoflurane significantly reduced posthemorrhagic ventricular dilation and improved motor, but not cognitive, functions in GMH animals 3 weeks after surgery; no improvements were observed after VPC23019 administration. CONCLUSIONS: Isoflurane post-treatment improved the neurological sequelae after GMH possibly by activation of the sphingosine kinase/Akt pathway.
Assuntos
Encéfalo/efeitos dos fármacos , Hemorragias Intracranianas/tratamento farmacológico , Isoflurano/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Modelos Animais de Doenças , Hemorragias Intracranianas/induzido quimicamente , Hemorragias Intracranianas/metabolismo , Isoflurano/farmacologia , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptores de Lisoesfingolipídeo/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Esfingosina/uso terapêuticoRESUMO
Treatments that could extend the therapeutic window of opportunity for stroke patients are urgently needed. Early administration of hyperbaric oxygen therapy (HBOT) has been proven neuroprotective in the middle cerebral artery occlusion (MCAo) in rodents. Our aim was to determine: 1) whether delayed HBOT after permanent MCAo (pMCAo) can still convey neuroprotection and restorative cell proliferation, and 2) whether these beneficial effects rely on HBO-induced activation of protein phosphatase-1γ (PP1-γ) leading to a decreased phosphorylation and ubiquitination of CREB and hence its stabilization. The experiments were performed in one hundred thirty-two male Sprague-Dawley rats with the body weight ranging from 240 to 270 g. Permanent MCAo was induced with the intraluminal filament occluding the right middle cerebral artery (MCA). In the first experiment, HBOT (2.5 ATA, 1h daily for 10 days) was started 48 h after pMCAo. Neurobehavioral deficits and infarct size as well as cyclic AMP response element-binding protein (CREB) expression and BrdU-DAB staining in the hippocampus and the peri-infarct region were evaluated on day 14 and day 28 post-MCAo. In the second experiment, HBOT (2.5 ATA, 1h) was started 3h after pMCAo. The effects of CREB siRNA or PP1-γ siRNA on HBO-induced infarct size alterations and target protein expression were studied. HBOT started with 48 h delay reduced infarct size, ameliorated neurobehavioral deficits and increased protein expression of CREB, resulting in increased cell proliferations in the hippocampus and peri-infarct region, on day 14 and day 28 post-MCAo. In the acute experiment pMCAo resulted in cerebral infarction and functional deterioration and reduced brain expression of PP1-γ, which led to increased phosphorylation and ubiquitination of CREB 24h after MCAo. However HBOT administered 3h after ischemia reversed these molecular events and resulted in CREB stabilization, infarct size reduction and neurobehavioral improvement. Gene silencing with CREB siRNA or PP1-γ siRNA reduced acute beneficial effects of HBO. In conclusion, delayed daily HBOT presented as potent neuroprotectant in pMCAo rats, increased CREB expression and signaling activity, and bolstered regenerative type cell proliferation in the injured brain. As shown in the acute experiment these effects of HBO were likely to be mediated by reducing ubiquitin-dependent CREB degradation owing to HBO-induced activation of PP1γ.
Assuntos
Proteína de Ligação a CREB/metabolismo , Oxigenoterapia Hiperbárica/métodos , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/terapia , Animais , Western Blotting , Proliferação de Células , Modelos Animais de Doenças , Imuno-Histoquímica , Imunoprecipitação , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND AND PURPOSE: Perihematomal edema formation and consequent cell death contribute to the delayed brain injury evoked by intracerebral hemorrhage (ICH). We aimed to evaluate the effect of α7 nicotinic acetylcholine receptor (α7nAChR) stimulation on behavior, brain edema, and neuronal apoptosis. Furthermore, we aimed to determine the role of the proapoptotic glycogen synthase kinase-3ß (GSK-3ß) after experimental ICH. METHODS: Male CD-1 mice (n=109) were subjected to intracerebral infusion of autologous blood (n=88) or sham surgery (n=21). ICH animals received vehicle administration, 4 or 12 mg/kg of α7nAChR agonist PHA-543613, 12 mg/kg of α7nAChR agonist PNU-282987, 6 mg/kg of α7nAChR antagonist methyllycaconitine (MLA), 15 µg/kg of phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, or PHA-543613 combined with MLA or wortmannin. Behavioral deficits and brain water content were evaluated at 24 and 72 hours after surgery. Western blotting and immunofluorescence staining were used for the quantification and localization of activated Akt (p-Akt), GSK-3ß (p-GSK-3ß), and cleaved caspase-3 (CC3). Neuronal cell death was quantified through terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). RESULTS: α7nAChR stimulation improved neurological outcome and reduced brain edema at 24 and 72 hours after surgery (P<0.05 compared with vehicle). Furthermore, PHA-543613 treatment increased p-Akt and decreased p-GSK-3ß and CC3 expressions in the ipsilateral hemisphere (P<0.05, respectively), which was reversed by MLA and wortmannin. P-Akt, p-GSK-3ß, and CC3 were generally localized in neurons. PHA-543613 reduced neuronal cell death in the perihematomal area (P<0.05). CONCLUSIONS: α7nAChR stimulation improved functional and morphological outcomes after experimental ICH in mice. PHA-543613 reduced the expression of proapoptotic GSK-3ß through the PI3K-Akt signaling pathway.
Assuntos
Benzamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Hemorragias Intracranianas/tratamento farmacológico , Fármacos Neuroprotetores , Agonistas Nicotínicos/farmacologia , Quinuclidinas/farmacologia , Receptores Nicotínicos/fisiologia , Animais , Apoptose/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Western Blotting , Água Corporal/metabolismo , Química Encefálica/efeitos dos fármacos , Edema Encefálico/etiologia , Edema Encefálico/patologia , Caspase 3/fisiologia , Inibidores de Caspase , Imunofluorescência , Glicogênio Sintase Quinase 3 beta , Hemorragias Intracranianas/psicologia , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Receptores Nicotínicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/psicologia , Resultado do Tratamento , Receptor Nicotínico de Acetilcolina alfa7RESUMO
BACKGROUND AND PURPOSE: Erythropoietin (EPO) has been demonstrated to possess significant neuroprotective effects in stroke. We determined if the nano-drug form of human recombinant EPO (PLGA-EPO nanoparticles [PLGA-EPO-NP]) can enhance neuroprotection at lower dosages versus human recombinant EPO (r-EPO). METHODS: Established neonatal rat model of unilateral ischemic stroke was used to compare r-EPO, PLGA-EPO-NP and phosphate-buffered saline, given by daily intraperitoneal injections, followed by infarction volume and Rotarod Performance Test assessment. RESULTS: PLGA-EPO-NP significantly reduced infarction volumes 72 hours after injury compared with the same concentrations of r-EPO. Functional deficits were significantly reduced by 300 U/kg PLGA-EPO-NP versus controls, with deficit attenuation apparent at significantly lower dosages of PLGA-EPO-NP versus r-EPO. CONCLUSIONS: PLGA-EPO-NP is neuroprotective and beneficial against deficits after brain ischemia, at significantly reduced dosages versus r-EPO.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Eritropoetina/farmacologia , Hipóxia Encefálica/tratamento farmacológico , Fármacos Neuroprotetores , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Isquemia Encefálica/patologia , Isquemia Encefálica/psicologia , Infarto Cerebral/tratamento farmacológico , Infarto Cerebral/patologia , Infarto Cerebral/psicologia , Eritropoetina/administração & dosagem , Excipientes , Feminino , Hipóxia Encefálica/patologia , Hipóxia Encefálica/psicologia , Injeções Intraperitoneais , Ácido Láctico , Masculino , Nanopartículas , Doenças do Sistema Nervoso/prevenção & controle , Doenças do Sistema Nervoso/psicologia , Tamanho do Órgão/fisiologia , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Equilíbrio Postural/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Teste de Desempenho do Rota-RodRESUMO
Fibroblast growth factors (FGFs) maintain and promote vascular integrity; however whether FGFs protect the blood-brain barrier (BBB) after intracerebral hemorrhage (ICH) remains unexplored. In this present study, we hypothesized that exogenous FGF administration attenuates brain injury after ICH, specifically by preserving endothelial adherens junctions, therefore reducing vasogenic brain edema and attenuating neurofunctional deficits in mice subjected to experimental ICH. Acid fibroblast growth factor (FGF1) or basic fibroblast growth factor (FGF2) was administered intracerebroventricularly (ICV) at 0.5 h after intrastriatal injection of bacterial collagenase (cICH) or autologous whole blood (bICH). Fibroblast growth factor receptor (FGFR) inhibitor PD173074 and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were additionally administered with FGF2. The selective Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) inhibitor Y27632 was independently administered at 0.5 h after cICH. Brain water content and neurofunctional deficits were evaluated at 24 and 72h after ICH induction. Evans blue extravasation as well as Western blot analysis for the quantification of activated FGFR, Akt, Ras-related C3 botulinum toxin substrate 1 (Rac1), Ras homolog gene family member A (RhoA) and adherens junction proteins (p120-catenin, ß-catenin and VE-cadherin) were conducted at 72 h post-cICH. FGF treatment reduced perihematomal brain edema and improved neurofunctional deficits at 72 h after experimental ICH (p<0.05, compared to vehicle); however, FGFR and PI3K inhibition reversed these neuroprotective effects. Exogenous FGF2 increased activated FGFR, Akt, and Rac1 but reduced activated RhoA protein expression at 72 h after cICH (p<0.05, compared to vehicle), which was reversed by FGFR and PI3K inhibition. Y27632 treatment reduced brain injury at 72 h after cICH (p<0.05, compared to vehicle) and increased the expression of catenins (p120-catenin, ß-catenin). In conclusion, our findings suggest that exogenous FGF treatment reduced RhoA activity via FGFR-induced activation of the PI3K-Akt-Rac1 signaling pathway, thus preserving BBB integrity, and therefore attenuating secondary brain injury after experimental ICH in mice.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Hemorragia Cerebral Traumática/tratamento farmacológico , Fator 1 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Animais , Barreira Hematoencefálica/fisiologia , Hemorragia Cerebral Traumática/metabolismo , Hemorragia Cerebral Traumática/fisiopatologia , Infusões Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos , Proteínas Recombinantes/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
BACKGROUND AND PURPOSE: Early brain injury is an important pathological process after subarachnoid hemorrhage (SAH). The goal of this study was to evaluate whether the α7 nicotinic acetylcholine receptor (α7nAChR) agonist PNU-282987 attenuates early brain injury after SAH and whether α7nAChR stimulation is associated with down-regulation of caspase activity via phosphatidylinositol 3-kinase-Akt signaling. METHODS: The perforation model of SAH was performed, and neurological score, body weight loss, and brain water content were evaluated 24 and 72 hours after surgery. Western blot and immunohistochemistry were used for quantification and localization of phosphorylated Akt and cleaved caspase 3. Neuronal cell death was quantified with TUNEL staining. α7nAChR antagonist methylcaconitine and phosphatidylinositol 3-kinase inhibitor wortmannin were used to manipulate the proposed pathway, and results were quantified with Western blot. RESULTS: PNU-282987 improved neurological deficits both 24 and 72 hours after surgery and reduced brain water content in left hemispheres 24 hours after surgery. PNU-282987 significantly increased phosphorylated Akt levels and significantly decreased cleaved caspase 3 levels in ipsilateral hemispheres after SAH. Methylcaconitine and wortmannin reversed effects of treatment. Phosphorylated Akt and cleaved caspase 3 were colocalized to neurons in the ipsilateral basal cortex. Phosphorylated Akt was mainly localized in TUNEL-negative cells. PNU-282987 significantly reduced neuronal cell death in the ipsilateral basal cortex. CONCLUSIONS: α7nAChR stimulation decreased neuronal cell death and brain edema and improved neurological status in a rat perforation model of SAH. α7nAChR stimulation is associated with increasing phosphorylation of Akt and decreasing cleaved caspase 3 levels in neurons.
Assuntos
Benzamidas/uso terapêutico , Lesões Encefálicas/tratamento farmacológico , Compostos Bicíclicos com Pontes/uso terapêutico , Agonistas Nicotínicos/uso terapêutico , Hemorragia Subaracnóidea/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Compostos Bicíclicos com Pontes/farmacologia , Caspase 3/metabolismo , Modelos Animais de Doenças , Frequência Cardíaca/efeitos dos fármacos , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Agonistas Nicotínicos/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/metabolismo , Hemorragia Subaracnóidea/patologiaRESUMO
Intracerebral hemorrhage (ICH) accounts for 20% of all strokes and is the most devastating form across all stroke types. Lymphocytes have been shown to potentiate cerebral inflammation and brain injury after stroke. FTY720 (Fingolimod) is an immune-modulating drug that prevents the egress of peripheral lymphocytes from peripheral stores. We hypothesized that FTY720 would reduce peripheral circulating lymphocytes, resulting in reduced brain injury and improved functional outcomes. CD-1 mice were anesthetized and then injected with collagenase into the right basal ganglia. Animals were divided into three groups: sham, ICH+Vehicle, and ICH+FTY720, by the intra-peritoneal route at 1 h after ICH induction. Brain water content was measured at 24 and 72 h. Neurobehavioral tests included corner test, forelimb use asymmetry, paw placement, wire-hang test, beam balance test, and a Neuroscore. FTY720 significantly reduced brain edema and improved neurological function at all time points tested. Lymphocyte modulation with FTY720 is an effective neuroprotective strategy to reduce brain injury and promote functional recovery after ICH.
Assuntos
Hemorragia Cerebral/tratamento farmacológico , Hemorragia Cerebral/fisiopatologia , Imunossupressores/uso terapêutico , Propilenoglicóis/uso terapêutico , Esfingosina/análogos & derivados , Análise de Variância , Animais , Edema Encefálico/etiologia , Edema Encefálico/prevenção & controle , Hemorragia Cerebral/induzido quimicamente , Colagenases/efeitos adversos , Modelos Animais de Doenças , Cloridrato de Fingolimode , Lateralidade Funcional , Camundongos , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/prevenção & controle , Exame Neurológico , Desempenho Psicomotor/efeitos da radiação , Esfingosina/uso terapêutico , Fatores de TempoRESUMO
Accounting for high mortality and morbidity rates, intracerebral hemorrhage (ICH) remains one of the most detrimental stroke subtypes lacking a specific therapy. Neuroinflammation contributes to ICH-induced brain injury and is associated with unfavorable outcomes. This study aimed to evaluate whether α7 nicotinic acetylcholine receptor (α7nAChR) stimulation ameliorates neuroinflammation after ICH. Male CD-1 mice and Sprague-Dawley were subjected to intracerebral injection of autologous blood or bacterial collagenase. ICH animals received either α7nAChR agonist PHA-543613 alone or combined with α7nAChR antagonist methyllycaconitine (MLA) or Janus kinase 2 (JAK2) antagonist AG490. Neurobehavioral deficits were evaluated at 24 hours, 72 hours, and 10 weeks after ICH induction. Perihematomal expressions of JAK2, signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor-α (TNF-α), and myeloperoxidase (MPO) were quantified via Western blot. Histologic volumetric analysis of brain tissues was conducted after 10 weeks following ICH induction. PHA-543613 improved short-term neurobehavioral (sensorimotor) deficits and increased activated perihematomal JAK2 and STAT3 expressions while decreasing TNF-α and MPO expressions after ICH. MLA reversed these treatment effects. PHA-543613 also improved long-term neurobehavioral (sensorimotor, learning, and memory) deficits and ameliorated brain atrophy after ICH. These treatment effects were reduced by AG490. α7nAChR stimulation reduced neuroinflammation via activation of the JAK2-STAT3 pathway, thereby ameliorating the short- and long-term sequelae after ICH.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Hemorragia Cerebral/tratamento farmacológico , Inflamação/tratamento farmacológico , Janus Quinase 2/genética , Fator de Transcrição STAT3/genética , Receptor Nicotínico de Acetilcolina alfa7/uso terapêutico , Animais , Transfusão de Sangue Autóloga/métodos , Lesões Encefálicas/etiologia , Lesões Encefálicas/genética , Lesões Encefálicas/fisiopatologia , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Hemorragia Cerebral/complicações , Hemorragia Cerebral/genética , Hemorragia Cerebral/fisiopatologia , Colagenases/administração & dosagem , Modelos Animais de Doenças , Humanos , Inflamação/complicações , Inflamação/genética , Inflamação/fisiopatologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Peroxidase/genética , Quinuclidinas/administração & dosagem , Ratos , Fator de Necrose Tumoral alfa/genética , Tirfostinas/administração & dosagem , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
We aim to determine if direct thrombin inhibition by dabigatran will improve long-term brain morphological and neurofunctional outcomes and if potential therapeutic effects are dependent upon reduced PAR-1 stimulation and consequent mTOR activation. Germinal matrix haemorrhage was induced by stereotaxically injecting 0.3 U type VII-S collagenase into the germinal matrix of P7 rat pups. Animals were divided into five groups: sham, vehicle (5% DMSO), dabigatran intraperitoneal, dabigatran intraperitoneal + TFLLR-NH2 (PAR-1 agonist) intranasal, SCH79797 (PAR-1 antagonist) intraperitoneal, and dabigatran intranasal. Neurofunctional outcomes were determined by Morris water maze, rotarod, and foot fault evaluations at three weeks. Brain morphological outcomes were determined by histological Nissl staining at four weeks. Expression levels of p-mTOR/p-p70s6k at three days and vitronectin/fibronectin at 28 days were quantified. Intranasal and intraperitoneal dabigatran promoted long-term neurofunctional recovery, improved brain morphological outcomes, and reduced intracranial pressure at four weeks after GMH. PAR-1 stimulation tended to reverse dabigatran's effects on post-haemorrhagic hydrocephalus development. Dabigatran also reduced expression of short-term p-mTOR and long-term extracellular matrix proteins, which tended to be reversed by PAR-1 agonist co-administration. PAR-1 inhibition alone, however, did not achieve the same therapeutic effects as dabigatran administration.
Assuntos
Antitrombinas/uso terapêutico , Dabigatrana/uso terapêutico , Hidrocefalia/prevenção & controle , Hemorragias Intracranianas/tratamento farmacológico , Administração Intranasal , Animais , Animais Recém-Nascidos , Antitrombinas/administração & dosagem , Dabigatrana/administração & dosagem , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/patologia , Hidrocefalia/etiologia , Hidrocefalia/patologia , Hidrocefalia/fisiopatologia , Injeções Intraperitoneais , Hemorragias Intracranianas/complicações , Hemorragias Intracranianas/patologia , Hemorragias Intracranianas/fisiopatologia , Aprendizagem em Labirinto/efeitos dos fármacos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Pirróis/administração & dosagem , Pirróis/farmacologia , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , Receptor PAR-1/agonistas , Receptor PAR-1/antagonistas & inibidores , Teste de Desempenho do Rota-RodRESUMO
Neonatal brain hemorrhage (NBH) of prematurity is an unfortunate consequence of preterm birth. Complications result in shunt dependence and long-term structural changes such as posthemorrhagic hydrocephalus, periventricular leukomalacia, gliosis, and neurological dysfunction. Several animal models are available to study this condition, and many basic mechanisms, etiological factors, and outcome consequences, are becoming understood. NBH is an important clinical condition, of which treatment may potentially circumvent shunt complication, and improve functional recovery (cerebral palsy, and cognitive impairments). This review highlights key pathophysiological findings of the neonatal vascular-neural network in the context of molecular mechanisms targeting the posthemorrhagic hydrocephalus affecting this vulnerable infant population.
Assuntos
Hemorragia Cerebral/congênito , Hemorragia Cerebral/fisiopatologia , Recém-Nascido Prematuro , Rede Nervosa/irrigação sanguínea , Rede Nervosa/fisiopatologia , Hemorragia Cerebral/patologia , Líquido Cefalorraquidiano/metabolismo , Humanos , Recém-Nascido , Recém-Nascido Prematuro/metabolismoRESUMO
Early brain injury (EBI), following subarachnoid hemorrhage (SAH), comprises blood-brain barrier (BBB) disruption and consequent edema formation. Peripheral leukocytes can infiltrate the injured brain, thereby aggravating BBB leakage and neuroinflammation. Thus, anti-inflammatory pharmacotherapies may ameliorate EBI and provide neuroprotection after SAH. Cannabinoid type 2 receptor (CB2R) agonism has been shown to reduce neuroinflammation; however, the precise protective mechanisms remain to be elucidated. This study aimed to evaluate whether the selective CB2R agonist, JWH133 can ameliorate EBI by reducing brain-infiltrated leukocytes after SAH. Adult male Sprague-Dawley rats were randomly assigned to the following groups: sham-operated, SAH with vehicle, SAH with JWH133 (1.0mg/kg), or SAH with a co-administration of JWH133 and selective CB2R antagonist SR144528 (3.0mg/kg). SAH was induced by endovascular perforation, and JWH133 was administered 1h after surgery. Neurological deficits, brain water content, Evans blue dye extravasation, and Western blot assays were evaluated at 24h after surgery. JWH133 improved neurological scores and reduced brain water content; however, SR144528 reversed these treatment effects. JWH133 reduced Evans blue dye extravasation after SAH. Furthermore, JWH133 treatment significantly increased TGF-ß1 expression and prevented an SAH-induced increase in E-selectin and myeloperoxidase. Lastly, SAH resulted in a decreased expression of the tight junction protein zonula occludens-1 (ZO-1); however, JWH133 treatment increased the ZO-1 expression. We suggest that CB2R stimulation attenuates neurological outcome and brain edema, by suppressing leukocyte infiltration into the brain through TGF-ß1 up-regulation and E-selectin reduction, resulting in protection of the BBB after SAH.
Assuntos
Edema Encefálico/complicações , Edema Encefálico/tratamento farmacológico , Quimiotaxia de Leucócito/efeitos dos fármacos , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/fisiologia , Hemorragia Subaracnóidea/complicações , Fator de Crescimento Transformador beta1/biossíntese , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Edema Encefálico/fisiopatologia , Canfanos/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Agonistas de Receptores de Canabinoides/uso terapêutico , Antagonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Canabinoides/uso terapêutico , Selectina E/biossíntese , Selectina E/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Peroxidase/biossíntese , Peroxidase/efeitos dos fármacos , Pirazóis/farmacologia , Ratos , Receptor CB2 de Canabinoide/antagonistas & inibidores , Hemorragia Subaracnóidea/tratamento farmacológico , Fator de Crescimento Transformador beta1/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/biossíntese , Proteína da Zônula de Oclusão-1/efeitos dos fármacosRESUMO
Formation of brain edema after intracerebral hemorrhage (ICH) is highly associated with its poor outcome. However, the relationship between cerebral edema and behavioral deficits has not been thoroughly examined in the preclinical setting. Hence, this study aimed to evaluate the ability of common sensorimotor tests to predict the extent of brain edema in two mouse models of ICH. One hundred male CD-1 mice were subjected to sham surgery or ICH induction via intrastriatal injection of either autologous blood (30 µL) or bacterial collagenase (0.0375U or 0.075U). At 24 and 72 h after surgery, animals underwent a battery of behavioral tests, including the modified Garcia neuroscore (Neuroscore), corner turn test (CTT), forelimb placing test (FPT), wire hang task (WHT) and beam walking (BW). Brain edema was evaluated via the wet weight/dry weight method. Intrastriatal injection of autologous blood or bacterial collagenase resulted in a significant increase in brain water content and associated sensorimotor deficits (p<0.05). A significant correlation between brain edema and sensorimotor deficits was observed for all behavioral tests except for WHT and BW. Based on these findings, we recommend implementing the Neuroscore, CTT and/or FPT in preclinical studies of unilateral ICH in mice.