RESUMEN
Unilateral brain injury in neonates results in largely contralateral hand function in children. Most research investigating neurorehabilitation targets for movement recovery has focused on the effects of brain injury on descending motor systems, especially the corticospinal tract. However, a recent human study demonstrated that sensory tract injury may have larger effects on dexterity than motor tract injury. To validate that the sensory tract injury impairs dexterity, we modeled the most common site of sensory tract injury in neonates by targeting the thalamocortical tract. In the postnatal day 7 rats, we used three types of lesions to the thalamocortical tract: periventricular blood injection, photothrombotic lesion, and electrolytic lesion. To test the sensitivity and specificity of these techniques, viral tracers were injected into the primary sensory or motor cortex immediately after injury. Electrolytic lesions were the most specific and reproducible for inducing a lesion compared to the other two methods. Electrolytic lesions disrupted 63% of the thalamocortical tract, while sparing the adjacent corticospinal tract in the internal capsule. To measure the impact on dexterity, the cylinder exploration and pasta handling tests were used to test the changes of forelimb use at 8 weeks after injury when the rats reached maturity. Lesions to the thalamocortical tract were associated with a significant decrease in the use of the contralateral forelimb in the cylinder task, and the degree of impairment positively correlated with the degree of injury. Overall, specific sensory system lesions of the thalamocortical tract impair forelimb use, suggesting a key role for skilled movement.
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Background and Purpose- Lacunar strokes are subcortical infarcts with small size and high disability rates, largely due to injury of the corticospinal tract in the internal capsule (IC). Current rodent models of lacunar infarcts are created based on stereotactic coordinates. We tested the hypothesis that better understanding of the somatotopy of the IC and guiding the lesion with electrical stimulation would allow a more accurate lesion to the forelimb axons of the IC. Methods- We performed electrophysiological motor mapping and viral tracing to define the somatotopy of the IC of Sprague Dawley rats. For the lesion, we used an optrode, which contains an electrode to localize forelimb responses and an optical fiber to deliver light. The infarct was induced when light activated the photothrombotic agent Rose Bengal, which was administered systemically. Results- We found largely a separate distribution of the forelimb and hindlimb axons in the IC, both by microstimulation mapping and tract tracing. Microstimulation-guided IC lesions ablated the forelimb axons of the IC in rats and caused lasting forelimb impairments while largely preserving the hindlimb axons of the IC and surrounding gray matter. Conclusions- Stimulation guidance enabled selective and reproducible infarcts of the forelimb axons of the IC in rats. Visual Overview- An online visual overview is available for this article.
Asunto(s)
Axones/fisiología , Estimulación Eléctrica , Infarto/fisiopatología , Cápsula Interna/cirugía , Accidente Cerebrovascular/cirugía , Animales , Axones/patología , Modelos Animales de Enfermedad , Femenino , Miembro Anterior/fisiopatología , Miembro Anterior/cirugía , Miembro Posterior/patología , Miembro Posterior/fisiopatología , Cápsula Interna/fisiopatología , Actividad Motora/fisiología , Corteza Motora/fisiopatología , Corteza Motora/cirugía , Tractos Piramidales/fisiopatología , Tractos Piramidales/cirugía , Ratas Sprague-Dawley , Recuperación de la Función/fisiología , Accidente Cerebrovascular/fisiopatologíaRESUMEN
[This corrects the article DOI: 10.3389/fncir.2018.00028.].
RESUMEN
After injury to the corticospinal tract (CST) in early development there is large-scale adaptation of descending motor pathways. Some studies suggest the uninjured hemisphere controls the impaired forelimb, while others suggest that the injured hemisphere does; these pathways have never been compared directly. We tested the contribution of each motor cortex to the recovery forelimb function after neonatal injury of the CST. We cut the left pyramid (pyramidotomy) of postnatal day 7 rats, which caused a measurable impairment of the right forelimb. We used pharmacological inactivation of each motor cortex to test its contribution to a skilled reach and supination task. Rats with neonatal pyramidotomy were further impaired by inactivation of motor cortex in both the injured and the uninjured hemispheres, while the forelimb of uninjured rats was impaired only from the contralateral motor cortex. Thus, inactivation demonstrated motor control from each motor cortex. In contrast, physiological and anatomical interrogation of these pathways support adaptations only in the uninjured hemisphere. Intracortical microstimulation of motor cortex in the uninjured hemisphere of rats with neonatal pyramidotomy produced responses from both forelimbs, while stimulation of the injured hemisphere did not elicit responses from either forelimb. Both anterograde and retrograde tracers were used to label corticofugal pathways. There was no increased plasticity from the injured hemisphere, either from cortex to the red nucleus or the red nucleus to the spinal cord. In contrast, there were very strong CST connections to both halves of the spinal cord from the uninjured motor cortex. Retrograde tracing produced maps of each forelimb within the uninjured hemisphere, and these were partly segregated. This suggests that the uninjured hemisphere may encode separate control of the unimpaired and the impaired forelimbs of rats with neonatal pyramidotomy.
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Lateralidad Funcional/fisiología , Actividad Motora/fisiología , Corteza Motora/fisiopatología , Plasticidad Neuronal/fisiología , Tractos Piramidales/crecimiento & desarrollo , Tractos Piramidales/lesiones , Adaptación Fisiológica/fisiología , Animales , Animales Recién Nacidos , Axones/patología , Axones/fisiología , Miembro Anterior/fisiopatología , Corteza Motora/efectos de los fármacos , Corteza Motora/patología , Trastornos del Movimiento/etiología , Trastornos del Movimiento/patología , Trastornos del Movimiento/fisiopatología , Tractos Piramidales/patología , Tractos Piramidales/fisiopatología , Ratas Sprague-Dawley , Recuperación de la Función/fisiologíaRESUMEN
BACKGROUND: Acute hypoxic/ischemic insults to the forebrain, often resulting in significant cellular loss of the cortical parenchyma, are a major cause of debilitating injury in the industrialized world. A clearer understanding of the pro-death/pro-survival signaling pathways and their downstream targets is critical to the development of therapeutic interventions to mitigate permanent neurological damage. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that the transcriptional repressor ZEB1, thought to be involved in regulating the timing and spatial boundaries of basic-Helix-Loop-Helix transactivator-mediated neurogenic determination/differentiation programs, functions to link a pro-survival transcriptional cascade rapidly induced in cortical neurons in response to experimentally induced ischemia. Employing histological, tissue culture, and molecular biological read-outs, we show that this novel pro-survival response, initiated through the rapid induction of p63, is mediated ultimately by the transcriptional repression of a pro-apoptotic isoform of p73 by ZEB1. We show further that this phylogenetically conserved pathway is induced as well in the human cortex subjected to episodes of clinically relevant stroke. CONCLUSIONS/SIGNIFICANCE: The data presented here provide the first evidence that ZEB1 induction is part of a protective response by neurons to ischemia. The stroke-induced increase in ZEB1 mRNA and protein levels in cortical neurons is both developmentally and phylogenetically conserved and may therefore be part of a fundamental cellular response to this insult. Beyond the context of stroke, the finding that ZEB1 is regulated by a member of the p53 family has implications for cell survival in other tissue and cellular environments subjected to ischemia, such as the myocardium and, in particular, tumor masses.
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Isquemia Encefálica/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Neuronas/citología , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Secuencia de Bases , Isquemia Encefálica/inducido químicamente , Muerte Celular , Hipoxia de la Célula , Supervivencia Celular , Células Cultivadas , Glucosa/deficiencia , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Oxígeno , Isoformas de Proteínas/metabolismo , Ratas , Accidente Cerebrovascular/inducido químicamente , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína Tumoral p73 , Regulación hacia Arriba , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Neonatal stroke is increasingly recognized in preterm and term infants, and the rate of arterial ischemic infarction occurring around the time of birth is as high as the annual incidence of large-vessel ischemic stroke in adults. Thus, neonatal stroke is a major contributor to perinatal morbidity and mortality, and a considerable number of these children will develop long-term neurodevelopmental disabilities. Our ability to investigate this situation has been limited by the technical challenges in developing suitable animal models. Our objective is to describe recent evidence in relation to animal models of neonatal stroke. In addition, we review and report potential neuroprotective strategies specific to neonatal stroke, with a focus on erythropoietin and cardiotrophin-1 because of their potential role in protection as well as repair.
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Citocinas/uso terapéutico , Eritropoyetina/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Proteínas del Tejido Nervioso/metabolismo , Factores de Riesgo , Accidente Cerebrovascular/epidemiología , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/fisiopatología , Factores de TiempoRESUMEN
Red ginseng root (Panax Ginseng CA Meyer) has been used clinically by many Asian people for thousands of years without any detrimental effects. One of the major components of Red ginseng root is ginsenoside Rb(1) (gRb1). Previously, we showed that intravenous infusion of gRb1 ameliorated ischemic brain damage through upregulation of an anti-apoptotic factor, Bcl-x(L) and that topical application of gRb1 to burn wound lesion facilitated wound healing through upregulation of vascular endothelial growth factor (VEGF). In the present study, we produced dihydroginsenoside Rb1 (dgRb1), a stable chemical derivative of gRb1, and showed that intravenous infusion of dgRb1 improved spinal cord injury (SCI) as well as ischemic brain damage. As we expected, the effective dose of dgRb1 was ten times lower than that of gRb1. Intravenous infusion of dgRb1 at this effective dose did not affect brain temperature, blood pressure or cerebral blood flow, suggesting that dgRb1 rescued damaged neurons without affecting systemic parameters. In subsequent in vitro studies that focused on dgRb1-induced expression of gene products responsible for neuronal death or survival, we showed that dgRb1 could upregulate the expression of not only Bcl-x(L), but also a potent angiogenic and neurotrophic factor, VEGF. We also showed that dgRb1-induced expression of bcl-x(L) and VEGF mRNA was HRE (hypoxia response element) and STRE (signal transducers and activators of transcription 5 (Stat5) response element) dependent, respectively.
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Infarto Encefálico/tratamiento farmacológico , Isquemia Encefálica/tratamiento farmacológico , Ginsenósidos/farmacología , Compresión de la Médula Espinal/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/genética , Proteína bcl-X/genética , Animales , Infarto Encefálico/fisiopatología , Infarto Encefálico/prevención & control , Isquemia Encefálica/genética , Isquemia Encefálica/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ginsenósidos/síntesis química , Ginsenósidos/uso terapéutico , Infusiones Intravenosas , Masculino , Estructura Molecular , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Factor de Transcripción STAT5/metabolismo , Compresión de la Médula Espinal/genética , Compresión de la Médula Espinal/fisiopatología , Resultado del Tratamiento , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
BACKGROUND/AIMS: Glucocorticoid receptors (GR) mediate cellular processes which may be neuroprotective and/or neurotoxic to the neonatal rat brain. Our aim was to describe GR ontogeny in the developing rat brain cortex and changes in GR expression after permanent neonatal focal cerebral ischemia (FCI). METHODS: GR Western blots and immunohistochemical stains were performed on neonatal rat cortices on P1, P3, P7, P10, P15, and P30 and on P7 at 1 h, 3 h, 6 h, 12 h, 24 h, and 72 h after FCI or sham-operation (S-O), 8 per group. Nissl staining was performed on FCI or S-O P7 cortical samples. RESULTS: Cortical GR expression was increased by 65.2% at P7, 110.1% at P15, and 87.0% at P30, compared to P1. On P7, GR expression decreased in the ischemic cortex after 6 h and in the non-ischemic cortex after 24 h of FCI (p < 0.05). Cortical GR expression was not altered in S-O P7 rats. Immunohistochemistry supported Western blot findings. Nissl staining revealed no gross decrease in neuronal number in non-ischemic cortices after 24 h of FCI, compared to baseline. CONCLUSIONS: Neonatal rat cortical GR expression increases during P1 to P30, peaking at P15. At P7, cortical GR expression appears downregulated in the ischemic cortex after 6 h and in the non-ischemic cortex after 24 h of FCI. Thus, cortical GR may play important roles in normal brain development and neonatal brain injury responses.
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Isquemia Encefálica/metabolismo , Corteza Cerebral/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Animales Recién Nacidos , Isquemia Encefálica/inducido químicamente , Isquemia Encefálica/patología , Corteza Cerebral/citología , Corteza Cerebral/embriología , Femenino , Inmunohistoquímica , Ratas , Ratas Sprague-DawleyRESUMEN
Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb(1) (gRb(1)) (C(54)H(92)O(23), molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb(1)-induced expression of gene products responsible for neuronal death or survival, we showed that gRb(1) stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-x(L) in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb(1) treatment. Ginsenoside Rb(1) appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.
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Isquemia Encefálica/patología , Muerte Celular/fisiología , Ginsenósidos/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Panax/química , Proteína bcl-X/metabolismo , Animales , Conducta Animal , Presión Sanguínea/fisiología , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Gerbillinae , Ginsenósidos/química , Humanos , Infarto de la Arteria Cerebral Media , Masculino , Aprendizaje por Laberinto , Estructura Molecular , Neuronas/metabolismo , Fármacos Neuroprotectores/química , Óxido Nítrico/metabolismo , Ratas , Ratas Endogámicas SHR , Factor de Transcripción STAT5/metabolismo , Proteína bcl-X/genéticaRESUMEN
Cardiotrophin-1 (CT-1) was initially defined as a mediator of cardiomyocyte hypertrophy. Additional studies have showed that CT-1 enhanced survival of differentiated cardiac muscle cells and inhibited cardiac myocyte apoptosis after serum deprivation or cytokine stimulation. Moreover, CT-1 has recently been shown to act as a neuroregulatory cytokine in the peripheral nervous system. However, its effects in the central nervous system have not been determined. In the present study, we evaluated whether CT-1 protects cultured cortical neurons against oxidative injuries caused by the hydroxyl radical-producing agent FeSO4 and by the peroxynitrite-producing agent 3-morpholinosydnonimine (SIN-1). CT-1 reduced neuronal cell death caused by FeSO4 and also attenuated the neurotoxic effect of SIN-1 in a dose-dependent manner. These results indicate that CT-1 is neuroprotective in an in vitro model of cerebral ischemia. This study indicates that further evaluation of CT-1 in acute brain injury should be investigated in vivo.
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Daño Encefálico Crónico/tratamiento farmacológico , Corteza Cerebral/efectos de los fármacos , Citocinas/farmacología , Radicales Libres/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Daño Encefálico Crónico/fisiopatología , Daño Encefálico Crónico/prevención & control , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatología , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Infarto Cerebral/tratamiento farmacológico , Infarto Cerebral/fisiopatología , Infarto Cerebral/prevención & control , Relación Dosis-Respuesta a Droga , Compuestos Férricos/efectos adversos , Compuestos Férricos/antagonistas & inhibidores , Depuradores de Radicales Libres/farmacología , Radicales Libres/efectos adversos , Molsidomina/efectos adversos , Molsidomina/análogos & derivados , Molsidomina/antagonistas & inhibidores , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/fisiología , RatasRESUMEN
We previously demonstrated that intermittent hypoxia evokes persistent changes in extracellular striatal dopamine, locomotor activity and executive function, using a rodent model emulating apnea of prematurity in which rat pups are exposed to 20-second bursts of hypoxic gas mix containing 10% oxygen (60 events/h; 6 h/day) from postnatal days 7 to 11. To determine whether subtle repetitive hypoxic insults also induce expression of stress-related genes, we employed real-time RT-PCR to assay gene transcription in neonatal rats subjected to the same paradigm. In addition, we also measured expression of stress-induced transcripts in an age-matched cohort following a more severe oxidative stressor: permanent focal ischemia. Four transcripts were elevated following the ischemic insult: heat shock protein 70 (Hsp70), CL100, nurr77, and heme oxygenase-1. In contrast, these transcripts were not regulated in the majority of neonatal rats exposed to an intermittent hypoxia protocol. Hsp70 was strongly induced, and CL100 and nurr77 were slightly induced in only 2 of 11 post-hypoxic rats compared to controls. These data demonstrate that a single ischemic event elicits expression of specific stress-related genes, whereas 5 days of brief intermittent hypoxic insults typically do not. Thus, it is unlikely that the neurochemical and behavioral morbidity observed in juvenile and adult rodents exposed to intermittent hypoxia during a critical period of brain development are related to stress-induced changes in gene expression.
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Encéfalo/metabolismo , Expresión Génica , Hipoxia/metabolismo , Estrés Oxidativo/genética , Animales , Animales Recién Nacidos , Proteínas de Ciclo Celular/genética , Fosfatasa 1 de Especificidad Dual , Femenino , Proteínas HSP70 de Choque Térmico/genética , Hemo-Oxigenasa 1/genética , Proteínas Inmediatas-Precoces/genética , Ataque Isquémico Transitorio/metabolismo , Masculino , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/análisis , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Perinatal brain injury is a major contributor to perinatal morbidity and mortality, and a considerable number of these children will develop long term neurodevelopmental disabilities. Despite the severe clinical and socio-economic significance and the advances in neonatal care over the past twenty years, no therapy yet exists that effectively prevents or ameliorates detrimental neurodevelopmental effects in cases of perinatal/neonatal brain injury. Our objective is to review recent evidence in relation to the pervading hypothesis for targeting time-dependent molecular and cellular repair mechanisms in the developing brain. In addition we review several potential neuroprotective strategies specific to the developing nervous system, with a focus on erythropoietin (Epo) because of its potential role in protection as well as repair.
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Encéfalo/embriología , Eritropoyetina/fisiología , Animales , Antioxidantes/metabolismo , Apoptosis , Encéfalo/metabolismo , Lesiones Encefálicas/patología , Isquemia Encefálica/patología , Radicales Libres , Humanos , Modelos Biológicos , Neuronas/metabolismo , Oxidantes/metabolismo , Receptores de Eritropoyetina , Cicatrización de HeridasRESUMEN
Erythropoietin (Epo) plays a central role in erythropoiesis but also has neuroprotective properties. Recently, Epo-related neuroprotective studies used a hypoxic-ischemic neonatal model, which is different from focal stroke, a frequent cause of neonatal brain injury. We report on the effects of Epo treatment given after focal stroke and its potential neuroprotective mechanisms in postnatal day 7 rats with focal cerebral ischemia (FCI) achieved by occlusion of the middle cerebral artery. The experimental groups included sham operation, FCI plus vehicle, and FCI plus Epo. In the Epo-treated group, pups received a single intraperitoneal injection of 1000 U/kg 15 min after FCI or three injections of 100, 1000, or 5000 U/kg, starting at 15 min and repeated at 1 and 2 d after FCI. Epo treatment produced significant reductions in the mean infarct area and volume at 1 and 3 d after FCI, demonstrated by 2,3,5-triphenyltetrazolium chloride staining. Terminal deoxynucleotidyltransferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining showed a markedly reduced number of TUNEL-positive cells in the Epo-treated group when compared with the vehicle control 3 d after FCI (p<0.01). The most effective dose after FCI was 1000 U/kg for 3 d. Immunoanalyses showed that Epo induced a significant increase in phosphorylated Janus kinase 2 and signal transducer and activator of transcription-5 expressions at 1 and 3 d and up-regulated Bcl-xL expression by 24 h after FCI but did not affect Epo receptor or NF-kappaB expression. In conclusion, Epo given after FCI in neonatal rats provides significant neuroprotection, mediated possibly by activation of the Janus kinase-signal transducer and activator of transcription-Bcl-xL signaling pathways.
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Isquemia Encefálica/tratamiento farmacológico , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/patología , Proteínas de Unión al ADN/metabolismo , Eritropoyetina/farmacología , Proteínas de la Leche/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Animales , Animales Recién Nacidos , Isquemia Encefálica/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Femenino , Etiquetado Corte-Fin in Situ , Infarto de la Arteria Cerebral Media , Janus Quinasa 2 , FN-kappa B/metabolismo , Embarazo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Eritropoyetina/metabolismo , Factor de Transcripción STAT5 , Proteína bcl-XRESUMEN
Erythropoietin (Epo) is a hematopoietic factor, which stimulates proliferation and differentiation of erythroid precursor cells. Epo also functions as a neuroprotective factor and protects neurons from ischemic damage. Recently a 17-mer peptide sequence (Epopeptide AB) in Epo (AEHCSLNENITVPDTKV) with a neuroprotective function was reported. In this study, we showed in vivo evidence that Epopeptide AB protected neurons from ischemic damage at similar dose compared to Epo. Epopeptide AB could not stimulate the proliferation of Epo-dependent growing murine myeloid Ep-FDC-P2 cells and also did not compete the proliferative function of Epo on these cells. Together with these results, Epopeptide AB did not transduce signals through direct binding to the known Epo receptor on hematopoietic cells but has neuroprotective activity against ischemia.
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Recent data suggest that the incidence of focal cerebral ischemia (FCI) and stroke is higher than previously recognized and could account for a large proportion of brain lesions in the preterm and full term neonate. Therefore, it is critically important to develop an appropriate model of FCI in neonatal animals. We describe here a modified model of permanent FCI in rat pups at postnatal day-7 (P7). To produce permanent FCI, a suture embolus with different diameters (180-220 microm) was inserted into the left common carotid artery (CCA) of the pups with different weight (14-19 g). Then the suture embolus was advanced to the middle cerebral artery (MCA) to produce its occlusion. The success of vascular occlusion was evaluated by imaging the ischemic territory on serial brain sections with carbon black staining immediately after permanent FCI. The consistent cerebral infarction was confirmed by 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 h after permanent FCI. Terminal deoxynucleotidyltransferase-mediated 2'-deoxyuridine 5'-triphospate-biotin nick end labeling (TUNEL) staining showed cell death with TUNEL labeling in the ischemic areas, which is one of the features of apoptosis. The present model opens the way for advanced pathophysiological studies of FCI in neonates.
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Isquemia Encefálica/fisiopatología , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/fisiopatología , Procedimientos Quirúrgicos Vasculares/métodos , Animales , Animales Recién Nacidos , Apoptosis/fisiología , Isquemia Encefálica/patología , Carbono , Infarto Cerebral/patología , Infarto Cerebral/fisiopatología , Femenino , Etiquetado Corte-Fin in Situ , Infarto de la Arteria Cerebral Media/patología , Embarazo , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Técnicas de Sutura , Sales de TetrazolioRESUMEN
Erythropoietin (Epo) has been shown to act as a neurotrophic and neuroprotective factor via binding to its receptor (EpoR) which is activated in adult brains following hypoxia and ischemia. However, no evidence suggests that cerebral ischemia can activate EpoR in the neonatal brain. In the present study, the changes in EpoR expression were investigated using a modified model of permanent focal cerebral ischemia (FCI) in 7-day-old rat pups. Western blot analysis with an anti-rabbit EpoR antibody revealed a significant increase in the EpoR protein in the ischemic areas, starting from 6 to 12 h after FCI. Moreover, many EpoR-positive cells were detected in the ischemic areas from 12 h after FCI, and the positive cells were identified as neurons and microglia/macrophage but not astrocytes 24 h after FCI. Additionally, double staining with a red in situ apoptosis detection kit and the EpoR antibody indicated that EpoR-positive cells were in apoptotic cell death in the ischemic area. Therefore, these results suggest that EpoR is activated in the ischemic areas of neonatal rats and plays an important role in brain injury during development.
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Animales Recién Nacidos/metabolismo , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Receptores de Eritropoyetina/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Femenino , Embarazo , Ratas , Ratas Sprague-Dawley , Receptores de Eritropoyetina/biosíntesisRESUMEN
Erythropoietin (EPO) promotes neuronal survival after cerebral ischemia in vivo and after hypoxia in vitro. However, the mechanisms underlying the protective effects of EPO on ischemic/hypoxic neurons are not fully understood. The present in vitro experiments showed that EPO attenuated neuronal damage caused by chemical hypoxia at lower extracellular concentrations (10(- 4)-10(-2) U/ml) than were previously considered. Moreover, EPO at a concentration of 10(-3) U/ml up-regulated Bcl-xL mRNA and protein expressions in cultured neurons. Subsequent in vivo study focused on whether EPO rescued hippocampal CA1 neurons from lethal ischemic damage and up-regulated the expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils. EPO was infused into the cerebroventricles of gerbils immediately after 3 min of ischemia for 28 days. Infusion of EPO at a dose of 5 U/day prevented the occurrence of ischemia-induced learning disability. Subsequent light microscopic examinations showed that pyramidal neurons in the hippocampal CA1 field were significantly more numerous in ischemic gerbils infused with EPO (5 U/day) than in those receiving vehicle infusion. The same dose of EPO infusion caused significantly more intense expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils than did vehicle infusion. These findings suggest that EPO prevents delayed neuronal death in the hippocampal CA1 field, possibly through up-regulation of Bcl-xL, which is known to facilitate neuron survival.