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Brain microvascular endothelial cells (BMECs), an important component of the neurovascular unit, can promote angiogenesis and synaptic formation in ischaemic mice after brain parenchyma transplantation. Since the therapeutic efficacy of cell-based therapies depends on the extent of transplanted cell residence in the target tissue and cell migration ability, the delivery route has become a hot research topic. In this study, we investigated the effects of carotid artery transplantation of BMECs on neuronal injury, neurorepair, and neurological dysfunction in rats after cerebral ischaemic attack. Purified passage 1 endothelial cells (P1-BMECs) were prepared from mouse brain tissue. Adult rats were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min. Then, the rats were treated with 5 × 105 P1-BMECs through carotid artery infusion or tail vein injection. We observed that carotid artery transplantation of BMECs produced more potent neuroprotective effects than caudal injection in MCAO rats, including reducing infarct size and alleviating neurological deficits in behavioural tests. Carotid artery-transplanted BMECs displayed a wider distribution in the ischaemic rat brain. Immunostaining for endothelial progenitor cells and the mature endothelial cell markers CD34 and RECA-1 showed that carotid artery transplantation of BMECs significantly increased angiogenesis. Carotid artery transplantation of BMECs significantly increased the number of surviving neurons, decreased the cerebral infarction volume, and alleviated neurological deficits. In addition, we found that carotid artery transplantation of BMECs significantly enhanced ischaemia-induced hippocampal neurogenesis, as measured by doublecortin (DCX) and Ki67 double staining within 2 weeks after ischaemic injury. We conclude that carotid artery transplantation of BMECs can promote cerebral angiogenesis, neurogenesis, and neurological function recovery in adult rats after ischaemic stroke. Our results suggest that carotid injection of BMECs may be a promising new approach for treating acute brain injuries.
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AIMS: Sleep disorders are prevalent among stroke survivors and impede stroke recovery, yet they are still insufficiently considered in the management of stroke patients, and the mechanisms by which they occur remain unclear. There is evidence that boosting phasic GABA signaling with zolpidem during the repair phase improves stroke recovery by enhancing neural plasticity; however, as a non-benzodiazepine hypnotic, the effects of zolpidem on post-stroke sleep disorders remain unclear. METHOD: Transient ischemic stroke in male rats was induced with a 30-minute middle cerebral artery occlusion. Zolpidem or vehicle was intraperitoneally delivered once daily from 2 to 7 days after the stroke, and the electroencephalogram and electromyogram were recorded simultaneously. At 24 h after ischemia, c-Fos immunostaining was used to assess the effect of transient ischemic stroke and acute zolpidem treatment on neuronal activity. RESULTS: In addition to the effects on reducing brain damage and mitigating behavioral deficits, repeated zolpidem treatment during the subacute phase of stroke quickly ameliorated circadian rhythm disruption, alleviated sleep fragmentation, and increased sleep depth in ischemic rats. Immunohistochemical staining showed that in contrast to robust activation in para-infarct and some remote areas by 24 h after the onset of focal ischemia, the activity of the ipsilateral suprachiasmatic nucleus, the biological rhythm center, was strongly suppressed. A single dose of zolpidem significantly upregulated c-Fos expression in the ipsilateral suprachiasmatic nucleus to levels comparable to the contralateral side. CONCLUSION: Stroke leads to suprachiasmatic nucleus dysfunction. Zolpidem restores suprachiasmatic nucleus activity and effectively alleviates post-stroke sleep disturbances, indicating its potential to promote stroke recovery.
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Accidente Cerebrovascular Isquémico , Trastornos del Sueño-Vigilia , Accidente Cerebrovascular , Humanos , Masculino , Ratas , Animales , Zolpidem/farmacología , Zolpidem/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/tratamiento farmacológico , Trastornos del Sueño-Vigilia/tratamiento farmacológico , Trastornos del Sueño-Vigilia/etiología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Sueño , Accidente Cerebrovascular Isquémico/tratamiento farmacológicoRESUMEN
It has been demonstrated that diabetes cause neurite degeneration in the brain and cognitive impairment and neurovascular interactions are crucial for maintaining brain function. However, the role of vascular endothelial cells in neurite outgrowth and synaptic formation in diabetic brain is still unclear. Therefore, present study investigated effects of brain microvascular endothelial cells (BMECs) on high glucose (HG)-induced neuritic dystrophy using a coculture model of BMECs with neurons. Multiple immunofluorescence labelling and western blot analysis were used to detect neurite outgrowth and synapsis formation, and living cell imaging was used to detect uptake function of neuronal glucose transporters. We found cocultured with BMECs significantly reduced HG-induced inhibition of neurites outgrowth (including length and branch formation) and delayed presynaptic and postsynaptic development, as well as reduction of neuronal glucose uptake capacity, which was prevented by pre-treatment with SU1498, a vascular endothelial growth factor (VEGF) receptor antagonist. To analyse the possible mechanism, we collected BMECs cultured condition medium (B-CM) to treat the neurons under HG culture condition. The results showed that B-CM showed the same effects as BMEC on HG-treated neurons. Furthermore, we observed VEGF administration could ameliorate HG-induced neuronal morphology aberrations. Putting together, present results suggest that cerebral microvascular endothelial cells protect against hyperglycaemia-induced neuritic dystrophy and restorate neuronal glucose uptake capacity by activation of VEGF receptors and endothelial VEGF release. This result help us to understand important roles of neurovascular coupling in pathogenesis of diabetic brain, providing a new strategy to study therapy or prevention for diabetic dementia. Hyperglycaemia induced inhibition of neuronal glucose uptake and impaired to neuritic outgrowth and synaptogenesis. Cocultured with BMECs/B-CM and VEGF treatment protected HG-induced inhibition of glucose uptake and neuritic outgrowth and synaptogenesis, which was antagonized by blockade of VEGF receptors. Reduction of glucose uptake may further deteriorate impairment of neurites outgrowth and synaptogenesis.
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Células Endoteliales , Hiperglucemia , Humanos , Células Endoteliales/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Cultivadas , Neuronas/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular/farmacología , Encéfalo/metabolismo , Glucosa/toxicidad , Glucosa/metabolismoRESUMEN
Reactive astrocytes can be transformed into new neurons. Vascular endothelial growth factor (VEGF) promotes the transformation of reactive astrocytes into neurons in ischemic brain. Therefore, in this study, the molecular mechanism of VEGF's effect on ischemia/hypoxia-induced astrocyte to neuron transformation was investigated in the models of rat middle cerebral artery occlusion (MCAO) and in astrocyte culture with oxygen and glucose deprivation (OGD). We found that VEGF enhanced ischemia-induced Pax6, a neurogenic fate determinant, expression and Erk phosphorylation in reactive astrocytes and reduced infarct volume of rat brain at 3 days after MCAO, which effects could be blocked by administration of U0126, a MAPK/Erk inhibitor. In cultured astrocytes, VEGF also enhanced OGD-induced Erk phosphorylation and Pax6 expression, which was blocked by U0126, but not wortmannin, a PI3K/Akt inhibitor, or SB203580, a MAPK/p38 inhibitor, suggesting VEGF enhanced Pax6 expression via activation of MAPK/Erk pathway. OGD induced the increase of miR365 and VEGF inhibited the increase of OGD-induced miR365 expression. However, miR365 agonists blocked VEGF-enhanced Pax6 expression in hypoxic astrocytes, but did not block VEGF-enhanced Erk phosphorylation. We further found that VEGF promoted OGD-induced astrocyte-converted to neuron. Interestingly, both U0126 and Pax6 RNAi significantly reduced enhancement of VEGF on astrocytes-to-neurons transformation, as indicated Dcx and MAP2 immunopositive signals in reactive astrocytes. Moreover, those transformed neurons become mature and functional. We concluded that VEGF enhanced astrocytic neurogenesis via the MAPK/Erk-miR-365-Pax6 signal axis. The results also indicated that astrocytes play important roles in the reconstruction of neurovascular units in brain after stroke.
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Astrocitos , Factor A de Crecimiento Endotelial Vascular , Ratas , Animales , Astrocitos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Sistema de Señalización de MAP Quinasas , Transdiferenciación Celular , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Infarto de la Arteria Cerebral Media/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Neuronas/metabolismo , Glucosa/metabolismoRESUMEN
Angiogenic factors play an important role in protecting, repairing, and reconstructing vessels after ischemic stroke. In the brains of transient focal cerebral ischemic mice, we observed a reduction in infarct volume after the administration of Angiopoietin 2 (Angpt2), but whether this process is promoted by Angpt2-induced angiogenesis has not been fully elaborated. Therefore, this study explored the angiogenic activities, in reference to CD34 which is a marker of activated ECs and blood vessels, of cultured ECs in vitro and in ischemic damaged cerebral area in mice following Angpt2 administration. Our results demonstrate that Angpt2 administration (100 ng/mL) is neuroprotective by significantly increasing the CD34 expression in in vitro-cultured ECs, reducing the infarct volume and mitigating neuronal loss, as well as enhancing CD34+ vascular length and area. In conclusion, these results indicate that Angpt2 promotes repair and attenuates ischemic injury, and that the mechanism of this is closely associated with angiogenesis in the brain after stroke.
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Allergic asthma is a complex lung disease characterized by breathlessness, airway inflammation, and obstruction. Allergy and allergic rhinitis (AR) are the main triggers of asthma. Vitamin A is an important supplementary factor for the physiological activation of the immune system. In the present study, we investigated the effects of vitamin A on the exacerbation of allergic asthma symptoms. BALB/c mice were allocated to four groups. Asthma was created in two groups, and in the other two groups, rhinitis was induced. One of the asthma groups and one of the rhinitis groups orally received vitamin A (20 IU/g for 15 days). The levels of Immunoglobulin (Ig) E, histamine, leukotriene B4 (LTB4), Cysteinyl leukotriene receptor (Cys-LT), interleukin (IL)-4, IL-5, IL-13, and IL-35 as well as eosinophil peroxidase activity, were measured. Also, the histopathology of mice lungs was evaluated. The levels of total IgE, LTB4, Cys-LT, IL-4, IL-5, IL-17, and IL-33, eosinophil peroxidase activity, perivascular and peribronchial inflammation significantly decreased in vitamin A-treated asthma and rhinitis groups compared to non-treated groups. Also, IL-13 and histamine levels, hyperplasia of the goblet cell, and hyper-secretion of the mucus insignificantly decreased in vitamin A-treated asthma and rhinitis groups. Asthma and AR are common diseases that are generally developed due to the dysregulation of the immune system. Vitamin A plays an important role in controlling the immunopathologic mechanisms of allergic diseases. Vitamin A could be a useful supplement in managing AR and asthma by decreasing the severity of inflammatory responses. Therefore, control of vitamin A deficiency is recommended in Allergy.
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Antiinflamatorios/uso terapéutico , Asma/tratamiento farmacológico , Rinitis Alérgica/tratamiento farmacológico , Vitamina A/uso terapéutico , Vitaminas/uso terapéutico , Animales , Asma/inmunología , Asma/metabolismo , Asma/patología , Biomarcadores/metabolismo , Femenino , Ratones , Ratones Endogámicos BALB C , Gravedad del Paciente , Rinitis Alérgica/inmunología , Rinitis Alérgica/metabolismo , Rinitis Alérgica/patología , Resultado del TratamientoRESUMEN
BACKGROUND: Sleep disorders are highly prevalent among stroke survivors and impede stroke recovery. It is well established that melatonin has neuroprotective effects in animal models of ischemic stroke. However, as a modulator of endogenous physiological circadian rhythms, the effects of melatonin on poststroke sleep disorders remain unclear. In the present study, we investigated how melatonin delivered intraperitoneally once daily in the subacute phase after stroke onset, influencing neuronal survival, motor recovery, and sleep-wake profiles in rats. METHODS: Transient ischemic stroke in male Sprague-Dawley rats was induced with 30 min occlusion of the middle cerebral artery. Melatonin or vehicle was delivered intraperitoneally once daily in the subacute phase, from 2 to 7 days after stroke. Electroencephalogram and electromyogram recordings were obtained simultaneously. RESULTS: Compared to the effects observed in the vehicle-treated ischemic group, after 6 daily consecutive treatment of melatonin at 10 mg/kg starting at ischemic/reperfusion day 2, the infarct volume was significantly decreased (from 39.6 to 26.2%), and the degeneration of axons in the ipsilateral striatum and the contralateral corpus callosum were significantly alleviated. Sensorimotor performances were obviously improved as evidenced by significant increases in the latency to falling off the wire and in the use of the impaired forelimb. In addition to those predictable results of reducing brain tissue damage and mitigating behavioral deficits, repeated melatonin treatment during the subacute phase of stroke also alleviated sleep fragmentation through reducing sleep-wake stage transitions and stage bouts, together with increasing stage durations. Furthermore, daily administration of melatonin at 9 a.m. significantly increased the nonrapid eye movement sleep delta power during both the light and dark periods and decreased the degree of reduction of the circadian index. CONCLUSIONS: Melatonin promptly reversed ischemia-induced sleep disturbances. The neuroprotective effects of melatonin on ischemic injury may be partially associated with its role in sleep modulation.
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Melatonina , Trastornos del Sueño-Vigilia , Animales , Ritmo Circadiano , Suplementos Dietéticos , Isquemia , Masculino , Melatonina/farmacología , Ratas , Ratas Sprague-Dawley , SueñoRESUMEN
A traditional penetration test only measures the total penetration within 5 s. The penetration process is not monitored, and therefore, a large amount of information on the deformation properties of asphalt is not used. This paper documents a study to use a high-speed camera to quantify the entire penetration process and use the Finite Element Method (FEM) to interpret the penetration process using a viscoelastic model. The penetration-time relationships of several asphalt binders (70#, 90#, a rubber modified binder, and a styrene-butadiene-styrene (SBS) modified binder) have been acquired using the new method, and the FEM modeling of the penetration processes is performed. The results show that both stress relaxation and creep appear during the penetration process. The results indicate that the improved test method and its data interpretation procedure may better characterize the properties of asphalt binder, which may extend the applications of the traditional penetration test.
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Pharmacological studies have indirectly shown that necroptosis participates in ischemic neuronal death. However, its mechanism has yet to be elucidated in the ischemic brain. TNFα-triggered RIPK1 kinase activation could initiate RIPK3/MLKL-mediated necroptosis under inhibition of caspase-8. In the present study, we performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia in rats and used immunoblotting and immunostaining combined with pharmacological analysis to study the mechanism of necroptosis in ischemic brains. In the ipsilateral hemisphere, we found that ischemia induced the increase of (i) RIPK1 phosphorylation at the Ser166 residue (p-RIPK1), representing active RIPK1 kinase and (ii) the number of cells that were double stained with P-RIPK1 (Ser166) (p-RIPK1+) and TUNEL, a label of DNA double-strand breaks, indicating cell death. Furthermore, ischemia induced activation of downstream signaling factors of RIPK1, RIPK3 and MLKL, as well as the formation of mature interleukin-1ß (IL-1ß). Treatment with necrostatin-1 (Nec-1), an inhibitor of necroptosis, significantly decreased ischemia-induced increase of p-RIPK1 expression and p-RIPK1+ neurons, which showed protection from brain damage. Meanwhile, Nec-1 reduced RIPK3, MLKL and p-MLKL expression levels and mature IL-1ß formation in Nec-1 treated ischemic brains. Our results clearly demonstrated that phosphorylation of RIPK1 at the Ser166 residue was involved in the pathogenesis of necroptosis in the brains after ischemic injury. Nec-1 treatment protected brains against ischemic necroptosis by reducing the activation of RIPK1 and inhibiting its downstream signaling pathways. These results provide direct in vivo evidence that phosphorylated RIPK1 (Ser 166) plays an important role in the initiation of RIPK3/MLKL-dependent necroptosis in the pathogenesis of ischemic stroke in the rodent brain.
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MicroRNA-365 (miR-365) is upregulated in the ischemic brain and is involved in oxidative damage in the diabetic rat. However, it is unclear whether miR-365 regulates oxidative stress (OS)-mediated neuronal damage after ischemia. Here, we used a transient middle cerebral artery occlusion model in rats and the hydrogen peroxide-induced OS model in primary cultured neurons to assess the roles of miR-365 in neuronal damage. We found that miR-365 exacerbated ischemic brain injury and OS-induced neuronal damage and was associated with a reduced expression of OXR1 (Oxidation Resistance 1). In contrast, miR-365 antagomir alleviated both the brain injury and OXR1 reduction. Luciferase assays indicated that miR-365 inhibited OXR1 expression by directly targeting the 3'-untranslated region of Oxr1. Furthermore, knockdown of OXR1 abolished the neuroprotective and antioxidant effects of the miR-365 antagomir. Our results suggest that miR-365 upregulation increases oxidative injury by inhibiting OXR1 expression, while its downregulation protects neurons from oxidative death by enhancing OXR1-mediated antioxidant signals.
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Antioxidantes/metabolismo , Isquemia Encefálica/metabolismo , MicroARNs/metabolismo , Proteínas Mitocondriales/metabolismo , Neuroprotección/fisiología , Estrés Oxidativo/fisiología , Animales , Isquemia Encefálica/prevención & control , Células Cultivadas , Técnicas de Silenciamiento del Gen/métodos , Peróxido de Hidrógeno/toxicidad , Masculino , MicroARNs/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
Astrocytic calcium signaling plays pivotal roles in the maintenance of neural functions and neurovascular coupling in the brain. Vascular endothelial growth factor (VEGF), an original biological substance of vessels, regulates the movement of calcium and potassium ions across neuronal membrane. In this study, we investigated whether and how VEGF regulates glutamate-induced calcium influx in astrocytes. We used cultured astrocytes combined with living cell imaging to detect the calcium influx induced by glutamate. We found that VEGF quickly inhibited the glutamate/hypoxia-induced calcium influx, which was blocked by an AMPA receptor antagonist CNQX, but not D-AP5 or UBP310, NMDA and kainate receptor antagonist, respectively. VEGF increased phosphorylation of PKCα and AMPA receptor subunit GluA2 in astrocytes, and these effects were diminished by SU1498 or calphostin C, a PKC inhibitor. With the pHluorin assay, we observed that VEGF significantly increased membrane insertion and expression of GluA2, but not GluA1, in astrocytes. Moreover, siRNA-produced knockdown of GluA2 expression in astrocytes reversed the inhibitory effect of VEGF on glutamate-induced calcium influx. Together, our results suggest that VEGF reduces glutamate-induced calcium influx in astrocytes via enhancing PKCα-mediated GluA2 phosphorylation, which in turn promotes the membrane insertion and expression of GluA2 and causes AMPA receptors to switch from calcium-permeable to calcium-impermeable receptors, thereby inhibiting astrocytic calcium influx. The present study reveals that excitatory neurotransmitter glutamate-mediated astrocytic calcium influx can be regulated by vascular biological factor via activation of AMPA receptor GluA2 subunit and uncovers a novel coupling mechanism between astrocytes and endothelial cells within the neurovascular unit.
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Astrocitos/metabolismo , Señalización del Calcio/fisiología , Proteína Quinasa C/metabolismo , Receptores AMPA/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Antagonistas de Aminoácidos Excitadores/farmacología , Ratas , Ratas Sprague-Dawley , Receptores AMPA/agonistas , Receptores AMPA/antagonistas & inhibidoresRESUMEN
Brain microvascular endothelial cells (BMEC) are highly complex regulatory cells that communicate with other cells in the neurovascular unit. Cerebral ischemic injury is known to produce detectable synaptic dysfunction. This study aims to investigate whether endothelial cells in the brain regulate postnatal synaptic development and to elucidate their role in functional recovery after ischemia. Here, we found that in vivo engraftment of endothelial cells increased synaptic puncta and excitatory postsynaptic currents in layers 2/3 of the motor cortex. This pro-synaptogenic effect was blocked by the depletion of VEGF in the grafted BMEC. The in vitro results showed that BMEC conditioned medium enhanced spine and synapse formation but conditioned medium without VEGF had no such effects. Moreover, under pathological conditions, transplanted endothelial cells were capable of enhancing angiogenesis and synaptogenesis and improved motor function in the ischemic injury model. Collectively, our findings suggest that endothelial cells promote excitatory synaptogenesis via the paracrine factor VEGF during postnatal development and exert repair functions in hypoxia-ischemic neonatal mice. This study highlights the importance of the endothelium-neuron interaction not only in regulating neuronal development but also in maintaining healthy brain function.
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Isquemia Encefálica/fisiopatología , Células Endoteliales/fisiología , Potenciales Postsinápticos Excitadores , Corteza Motora/irrigación sanguínea , Corteza Motora/crecimiento & desarrollo , Trastornos Motores/fisiopatología , Sinapsis/fisiología , Animales , Animales Recién Nacidos , Isquemia Encefálica/complicaciones , Células Cultivadas , Medios de Cultivo Condicionados , Femenino , Masculino , Ratones Endogámicos C57BL , Microvasos/fisiología , Trastornos Motores/etiología , Neovascularización Fisiológica , Tálamo/crecimiento & desarrollo , Factor A de Crecimiento Endotelial Vascular/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/fisiologíaRESUMEN
Reactive astrocytes induced by ischemia can transdifferentiate into mature neurons. This neurogenic potential of astrocytes may have therapeutic value for brain injury. Epigenetic modifications are widely known to involve in developmental and adult neurogenesis. PAX6, a neurogenic fate determinant, contributes to the astrocyte-to-neuron conversion. However, it is unclear whether microRNAs (miRs) modulate PAX6-mediated astrocyte-to-neuron conversion. In the present study we used bioinformatic approaches to predict miRs potentially targeting Pax6, and transient middle cerebral artery occlusion (MCAO) to model cerebral ischemic injury in adult rats. These rats were given striatal injection of glial fibrillary acidic protein targeted enhanced green fluorescence protein lentiviral vectors (Lv-GFAP-EGFP) to permit cell fate mapping for tracing astrocytes-derived neurons. We verified that miR-365 directly targets to the 3'-UTR of Pax6 by luciferase assay. We found that miR-365 expression was significantly increased in the ischemic brain. Intraventricular injection of miR-365 antagomir effectively increased astrocytic PAX6 expression and the number of new mature neurons derived from astrocytes in the ischemic striatum, and reduced neurological deficits as well as cerebral infarct volume. Conversely, miR-365 agomir reduced PAX6 expression and neurogenesis, and worsened brain injury. Moreover, exogenous overexpression of PAX6 enhanced the astrocyte-to-neuron conversion and abolished the effects of miR-365. Our results demonstrate that increase of miR-365 in the ischemic brain inhibits astrocyte-to-neuron conversion by targeting Pax6, whereas knockdown of miR-365 enhances PAX6-mediated neurogenesis from astrocytes and attenuates neuronal injury in the brain after ischemic stroke. Our findings provide a foundation for developing novel therapeutic strategies for brain injury.
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Astrocitos/metabolismo , MicroARNs/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Factor de Transcripción PAX6/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Antagomirs/administración & dosificación , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Hipoxia de la Célula/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Glucosa/deficiencia , Masculino , MicroARNs/antagonistas & inhibidores , Neuronas/patología , Ratas Sprague-Dawley , Accidente Cerebrovascular/patologíaRESUMEN
The objective of this study is to investigate whether the addition of double-filtration plasmapheresis (DFPP) to leflunomide and methotrexate repairs MRI bone erosion in patients with long-standing rheumatoid arthritis (RA). Seventy-two patients with highly active RA of > 3 years' duration were randomized to receive DFPP in addition to DMARDs (leflunomide and methotrexate) or DMARDs. Contrast-enhanced MRI of the right wrist was performed at months 0, 6, and 12. MRI bone erosion, synovitis, and bone edema were scored with validated methods. The primary endpoint was the change in MRI bone erosion over 12 months. Patients treated with DFPP in addition to DMARDs demonstrated significantly greater decrease in MRI erosion score compared with those treated with DMARDs, being 11.3 ± 9.6 at month 12, compared with 16.9 ± 8.3 in patients with DMARDs (P < 0.001), and compared with 14.4 ± 9.6 at baseline (P < 0.001). 84.2% of patients treated with DFPP in addition to DMARDs demonstrated a decrease in MRI erosion score. Synovitis and bone edema improved significantly with DFPP in addition to DMARDs compared with DMARDs at months 6 and 12. (1.05 ± 1.7 and 2.0 ± 3.9 compared with 8.0 ± 1.4 and 12.6 ± 7.9 at month 12). Patients without synovitis and bone edema reached in 55.3% among patients with DFPP in addition to DMARDs. This study demonstrated that DFPP combination therapy significantly decreased bone erosion and received the primary goal of repair of erosions through abrogating MRI inflammation (synovitis and bone edema) in long-standing RA patients with high disease activity. The findings suggest that addition of DFPP is associated with repair of erosions and further suppression of inflammation.
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Antirreumáticos/uso terapéutico , Artritis Reumatoide/diagnóstico por imagen , Isoxazoles/uso terapéutico , Articulaciones/diagnóstico por imagen , Imagen por Resonancia Magnética , Metotrexato/uso terapéutico , Plasmaféresis/métodos , Adulto , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/terapia , Terapia Combinada , Femenino , Humanos , Leflunamida , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Brain microvascular endothelial cells (BMEC) have been found to guide the migration, promote the survival and regulate the differentiation of neural cells. However, whether BMEC promote development and maturation of immature neurons is still unknown. Therefore, in this study, we used a direct endothelium-neuron co-culture system combined with patch clamp recordings and confocal imaging analysis, to investigate the effects of endothelial cells on neuronal morphology and function during development. We found that endothelial cells co-culture or BMEC-conditioned medium (B-CM) promoted neurite outgrowth and spine formation, accelerated electrophysiological development and enhanced synapse function. Moreover, B-CM treatment induced vascular endothelial growth factor (VEGF) expression and p38 phosphorylation in the cortical neurons. Through pharmacological analysis, we found that incubation with SU1498, an inhibitor of VEGF receptor, abolished B-CM-induced p-p38 upregulation and suppressed the enhancement of synapse formation and transmission. SB203580, an inhibitor of p38 MAPK also blocked B-CM-mediated synaptic regulation. Together these results clearly reveal that the endothelium-neuron interactions promote morphological and functional maturation of neurons. In addition, neurovascular interaction-mediated promotion of neural network maturation relies on activation of VEGF/Flk-1/p38 MAPK signaling. This study provides novel aspects of endothelium-neuron interactions and novel mechanism of neurovascular crosstalk.
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Vascular endothelial growth factor (VEGF) was originally recognized as a substance predominantly with vascular permeability and angiogenesis. Recently, more and more evidence indicated that VEGF is expressed in the neurons of the developing and adult brains. Functional investigation demonstrated that VEGF shows several important effects on the neuronal development and physiological function. For example, VEGF accelerates the development of neurons and neural dendritic and axon growth. Besides, VEGF directly and acutely regulates the functions of multiple ion channels of the neuron membrane and changes neural excitability. In traumatic or ischemic injured brains, VEGF produces neuroprotection, enhances capacity of adult neurogenesis and transformation of astroglial cells into new neurons, which are fundamental basis for re-establishment of neural network. Based on the knowledge obtained from the literatures, we propose that VEGF may play very important roles in neural plasticity in the normal brain, and the reconstruction of neurovascular units and neural repair in the traumatic injured brain. This review mainly focuses on neural activity and repair roles of VEGF in adult mammalian brains. Further study on the mechanism of VEGF's neurobiological effects in the brain will be helpful for understanding the regulation of brain functions and developing new therapeutic strategy for prevention of neurodegeneration of the brain.
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Astrocitos/citología , Lesiones Encefálicas/fisiopatología , Neurogénesis , Plasticidad Neuronal , Neuronas/citología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , HumanosRESUMEN
The present study focuses on the function of cpg15, a neurotrophic factor, in ischemic neuronal recovery using transient global cerebral ischemic (TGI) mouse model and oxygen-glucose deprivation (OGD)-treated primary cultured cells. The results showed that expression of cpg15 proteins in astrocytes, predominantly the soluble form, was significantly increased in mouse hippocampus after TGI and in the cultured astrocytes after OGD. Addition of the medium from the cpg15-overexpressed astrocytic culture into the OGD-treated hippocampal neuronal cultures reduces the neuronal injury, whereas the recovery of neurite outgrowths of OGD-injured neurons was prevented when cpg15 in the OGD-treated astrocytes was knocked down, or the OGD-treated-astrocytic medium was immunoadsorbed by cpg15 antibody. Furthermore, lentivirus-delivered knockdown of cpg15 expression in mouse hippocampal astrocytes diminishes the dendritic branches and exacerbates injury of neurons in CA1 region after TGI. In addition, treatment with inhibitors of MEK1/2, PI3K, and TrkA decreases, whereas overexpression of p-CREB, but not dp-CREB, increases the expression of cpg15 in U118 or primary cultured astrocytes. Also, it is observed that the Flag-tagged soluble cpg15 from the astrocytes transfected with Flag-tagged cpg15-expressing plasmids adheres to the surface of neuronal bodies and the neurites. In conclusion, our results suggest that the soluble cpg15 from astrocytes induced by ischemia could ameliorate the recovery of the ischemic-injured hippocampal neurons via adhering to the surface of neurons. The upregulated expression of cpg15 in astrocytes may be activated via MAPK and PI3K signal pathways, and regulation of CREB phosphorylation.SIGNIFICANCE STATEMENT Neuronal plasticity plays a crucial role in the amelioration of neurological recovery of ischemic injured brain, which remains a challenge for clinic treatment of cerebral ischemia. cpg15 as a synaptic plasticity-related factor may participate in promoting the recovery process; however, the underlying mechanisms are still largely unknown. The objective of this study is to reveal the function and mechanism of neuronal-specific cpg15 expressed in astrocytes after ischemia induction, in promoting the recovery of injured neurons. Our findings provided new mechanistic insight into the neurological recovery, which might help develop novel therapeutic options for cerebral ischemia via astrocytic-targeting interference of gene expression.
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Astrocitos/metabolismo , Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proyección Neuronal/fisiología , Neuronas/metabolismo , Animales , Astrocitos/patología , Isquemia Encefálica/patología , Línea Celular Tumoral , Células Cultivadas , Proteínas Ligadas a GPI/biosíntesis , Hipocampo/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuritas/metabolismo , Neuritas/patología , Neuronas/patología , Distribución Aleatoria , Recuperación de la Función/fisiología , SolubilidadRESUMEN
Axon-transport plays an important role in neuronal activity and survival. Reduced endogenous VEGF can cause neuronal damage and axon degeneration. It is unknown at this time if VEGF can be transported within the axon or whether it can be released by axonal depolarization. We transfected VEGF-eGFP plasmids in cultured hippocampal neurons and tracked their movement in the axons by live-cell confocal imaging. Then, we co-transfected phVEGF-eGFP and kinesin-1B-DsRed vectors into neurons and combined with immunoprecipitation and two-color imaging to study the mechanism of VEGF axon-trafficking. We found that VEGF vesicles morphologically co-localized and biochemically bounded with kinesin-1B, as well as co-trafficked with it in the axons. Moreover, the capacity for axonal trafficking of VEGF was reduced by administration of nocodazole, an inhibitor of microtubules, or kinesin-1B shRNA. In addition, we found that VEGF could release from the cultured neurons under acute depolarizing stimulation with potassium chloride. Therefore, present findings suggest that neuronal VEGF is stored in the vesicles, actively released, and transported in the axons, which depends on the presence of kinesin-1B and functional microtubules. These results further help us to understand the importance of neuronal VEGF in the maintenance of neuronal activity and survival throughout life.
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Abnormal expression and dysfunction of methyl-CpG binding protein 2 (MeCP2) cause Rett syndrome (RTT). The diverse phosphorylation modifications modulate MeCP2 function in neural cells. Using western blot and immunohistochemistry, we examined the expression patterns of MeCP2 and three phospho-MeCP2s (pMeCP2s) in the developing rat brain. The expression of MeCP2 and phospho-S80 (pS80) MeCP2 increased while pS421 MeCP2 and pS292 MeCP2 decreased with brain maturation. In contrast to the nuclear localization of MeCP2 and pS80 MeCP2, pS421 MeCP2 and pS292 MeCP2 were mainly expressed in the cytoplasmic compartment. Apart from their distribution in neurons, they were also detected at a low level in astrocytes. Postnatally-initiated MeCP2 deficiency affected cerebellar neural cell development, as determined by the abnormal expression of GFAP, DCX, Tuj1, MAP-2, and calbindin-D28k. Together, these results demonstrate that MeCP2 and diverse pMeCP2s have distinct features of spatio-temporal expression in the rat brain, and that the precise levels of MeCP2 in the postnatal period are vital to cerebellar neural cell development.
Asunto(s)
Encéfalo , Cerebelo/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteína 2 de Unión a Metil-CpG/metabolismo , Neuronas/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Proteína Doblecortina , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Masculino , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/inmunología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Monoéster Fosfórico Hidrolasas/farmacología , Fosforilación , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción Genética , Tubulina (Proteína)/metabolismoRESUMEN
This study examined the effect of neuron-endothelial coupling on the survival of neurons after ischemia and the possible mechanism underlying that effect. Whole-cell patch-clamp experiments were performed on cortical neurons cultured alone or directly cocultured with brain microvascular endothelial cells (BMEC). Propidium iodide (PI) and NeuN staining were performed to examine neuronal death following oxygen and glucose deprivation (OGD). We found that the neuronal transient outward potassium currents (IA) decreased in the coculture system, whereas the outward delayed-rectifier potassium currents (IK) did not. Sodium nitroprusside, a NO donor, enhanced BMEC-induced IA inhibition and nitro-l-arginine methylester, a NOS inhibitor, partially prevented this inhibition. Moreover, the neurons directly cocultured with BMEC showed more resistance to OGD-induced injury compared with the neurons cultured alone, and that neuroprotective effect was abolished by treatment with NS5806, an activator of the IA. These results indicate that vascular endothelial cells assist neurons to prevent hypoxic injury via inhibiting neuronal IA by production of NO in the direct neuron-BMEC coculture system. These results further provide direct evidence of functional coupling between neurons and vascular endothelial cells. This study clearly demonstrates that vascular endothelial cells play beneficial roles in the pathophysiological processes of neurons after hypoxic injury, suggesting that the improvement of neurovascular coupling or functional remodeling may become an important therapeutic target for preventing brain injury.