RESUMEN
Advances in our understanding of ADAMTS13 structure, and the conformation changes required for full activity, have rejuvenated the possibility of its use as a thrombolytic therapy. We have tested a novel Ala1144Val ADAMTS13 variant (constitutively active [ca] ADAMTS13) that exhibits constitutive activity, characterized using in vitro assays of ADAMTS13 activity, and greatly enhanced thrombolytic activity in 2 murine models of ischemic stroke, the distal FeCl3 middle cerebral artery occlusion (MCAo) model and transient middle cerebral artery occlusion (tMCAO) with systemic inflammation and ischemia/reperfusion injury. The primary measure of efficacy in both models was restoration of regional cerebral blood flow (rCBF) to the MCA territory, which was determined using laser speckle contrast imaging. The caADAMTS13 variant exhibited a constitutively active conformation and a fivefold enhanced activity against fluorescence resonance energy transfer substrate von Willebrand factor 73 (FRETS-VWF73) compared with wild-type (wt) ADAMTS13. Moreover, caADAMTS13 inhibited VWF-mediated platelet capture at subphysiological concentrations and enhanced t-PA/plasmin lysis of fibrin(ogen), neither of which were observed with wtADAMTS13. Significant restoration of rCBF and reduced lesion volume was observed in animals treated with caADAMTS13. When administered 1 hour after FeCl3 MCAo, the caADAMTS13 variant significantly reduced residual VWF and fibrin deposits in the MCA, platelet aggregate formation, and neutrophil recruitment. When administered 4 hours after reperfusion in the tMCAo model, the caADAMTS13 variant induced a significant dissolution of platelet aggregates and a reduction in the resulting tissue hypoperfusion. The caADAMTS13 variant represents a potentially viable therapeutic option for the treatment of acute ischemic stroke, among other thrombotic indications, due to its enhanced in vitro and in vivo activities that result from its constitutively active conformation.
Asunto(s)
Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Proteína ADAMTS13/genética , Animales , Antiinflamatorios/uso terapéutico , Fibrina , Fibrinolíticos/uso terapéutico , Infarto de la Arteria Cerebral Media/patología , Ratones , Accidente Cerebrovascular/tratamiento farmacológico , Factor de von Willebrand/uso terapéuticoRESUMEN
Background and Purpose- A major process contributing to cell death in the ischemic brain is inflammation. Inflammasomes are multimolecular protein complexes that drive inflammation through activation of proinflammatory cytokines, such as IL (interleukin)-1ß. Preclinical evidence suggests that IL-1ß contributes to a worsening of ischemic brain injury. Methods- Using a mouse middle cerebral artery thrombosis model, we examined the inflammatory response after stroke and the contribution of the NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome to ischemic injury. Results- There was a marked inflammatory response after stroke characterized by increased expression of proinflammatory cytokines and NLRP3 and by recruitment of leukocytes to the injured tissue. Targeting NLRP3 with the inhibitor MCC950, or using mice in which NLRP3 was knocked out, had no effect on the extent of injury caused by stroke. Conclusions- These data suggest that the NLRP3 pathway does not contribute to the inflammation exacerbating ischemic brain damage, contradicting several recent reports to the contrary.
Asunto(s)
Lesiones Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Trombosis Intracraneal/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/deficiencia , Accidente Cerebrovascular/metabolismo , Animales , Lesiones Encefálicas/patología , Isquemia Encefálica/patología , Furanos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos , Indenos , Inflamasomas/antagonistas & inhibidores , Inflamasomas/deficiencia , Trombosis Intracraneal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Accidente Cerebrovascular/patología , Sulfonamidas/farmacología , SulfonasRESUMEN
The cytokine interleukin-1 (IL-1) is a key contributor to neuroinflammation and brain injury, yet mechanisms by which IL-1 triggers neuronal injury remain unknown. Here we induced conditional deletion of IL-1R1 in brain endothelial cells, neurons and blood cells to assess site-specific IL-1 actions in a model of cerebral ischaemia in mice. Tamoxifen treatment of IL-1R1 floxed (fl/fl) mice crossed with mice expressing tamoxifen-inducible Cre-recombinase under the Slco1c1 promoter resulted in brain endothelium-specific deletion of IL-1R1 and a significant decrease in infarct size (29%), blood-brain barrier (BBB) breakdown (53%) and neurological deficit (40%) compared to vehicle-treated or control (IL-1R1fl/fl) mice. Absence of brain endothelial IL-1 signalling improved cerebral blood flow, followed by reduced neutrophil infiltration and vascular activation 24â¯h after brain injury. Conditional IL-1R1 deletion in neurons using tamoxifen inducible nestin-Cre mice resulted in reduced neuronal injury (25%) and altered microglia-neuron interactions, without affecting cerebral perfusion or vascular activation. Deletion of IL-1R1 specifically in cholinergic neurons reduced infarct size, brain oedema and improved functional outcome. Ubiquitous deletion of IL-1R1 had no effect on brain injury, suggesting beneficial compensatory mechanisms on other cells against the detrimental effects of IL-1 on endothelial cells and neurons. We also show that IL-1R1 signalling deletion in platelets or myeloid cells does not contribute to brain injury after experimental stroke. Thus, brain endothelial and neuronal (cholinergic) IL-1R1 mediate detrimental actions of IL-1 in the brain in ischaemic stroke. Cell-specific targeting of IL-1R1 in the brain could therefore have therapeutic benefits in stroke and other cerebrovascular diseases.
Asunto(s)
Isquemia Encefálica/inmunología , Interleucina-1/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Lesiones Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Neuronas Colinérgicas/metabolismo , Neuronas Colinérgicas/fisiología , Citocinas/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Inflamación/metabolismo , Interleucina-1/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Transducción de SeñalRESUMEN
Neutrophils are key components of the innate immune response, providing host defence against infection and being recruited to non-microbial injury sites. Platelets act as a trigger for neutrophil extravasation to inflammatory sites but mechanisms and tissue-specific aspects of these interactions are currently unclear. Here, we use bacterial endotoxin in mice to trigger an innate inflammatory response in different tissues and measure neutrophil invasion with or without platelet reduction. We show that platelets are essential for neutrophil infiltration to the brain, peritoneum and skin. Neutrophil numbers do not rise above basal levels in the peritoneum and skin and are decreased (~60%) in the brain when platelet numbers are reduced. In contrast neutrophil infiltration in the lung is unaffected by platelet reduction, up-regulation of CXCL-1 (2·4-fold) and CCL5 (1·4-fold) acting as a compensatory mechanism in platelet-reduced mice during lung inflammation. In brain inflammation targeting platelet receptor GPIbα results in a significant decrease (44%) in platelet-mediated neutrophil invasion, while maintaining platelet numbers in the circulation. These results suggest that therapeutic blockade of platelet GPIbα could limit the harmful effects of excessive inflammation while minimizing haemorrhagic complications of platelet reduction in the brain. The data also demonstrate the ability to target damaging brain inflammation in stroke and related disorders without compromising lung immunity and hence risk of pneumonia, a major complication post stroke. In summary, our data reveal an important role for platelets in neutrophil infiltration to various tissues, including the brain, and so implicate platelets as a key, targetable component of cerebrovascular inflammatory disease or injury.
Asunto(s)
Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Encéfalo/inmunología , Encéfalo/metabolismo , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Encéfalo/patología , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Modelos Animales de Enfermedad , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Lipopolisacáridos/inmunología , RatonesRESUMEN
Inflammation that contributes to acute cerebrovascular disease is driven by the proinflammatory cytokine interleukin-1 and is known to exacerbate resulting injury. The activity of interleukin-1 is regulated by multimolecular protein complexes called inflammasomes. There are multiple potential inflammasomes activated in diverse diseases, yet the nature of the inflammasomes involved in brain injury is currently unknown. Here, using a rodent model of stroke, we show that the NLRC4 (NLR family, CARD domain containing 4) and AIM2 (absent in melanoma 2) inflammasomes contribute to brain injury. We also show that acute ischemic brain injury is regulated by mechanisms that require ASC (apoptosis-associated speck-like protein containing a CARD), a common adaptor protein for several inflammasomes, and that the NLRP3 (NLR family, pyrin domain containing 3) inflammasome is not involved in this process. These discoveries identify the NLRC4 and AIM2 inflammasomes as potential therapeutic targets for stroke and provide new insights into how the inflammatory response is regulated after an acute injury to the brain.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Lesiones Encefálicas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Animales , Encéfalo/metabolismo , Encéfalo/patología , Isquemia Encefálica/patología , Proteínas Adaptadoras de Señalización CARD , Muerte Celular , Citocinas/metabolismo , Hipoxia/patología , Infarto de la Arteria Cerebral Media/patología , Inflamación/patología , Isquemia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Proteína con Dominio Pirina 3 de la Familia NLR , Estructura Terciaria de ProteínaRESUMEN
Neuroprotective strategies for ischemic stroke have failed to translate from bench to bedside, possibly due to the lack of consideration of key clinical co-morbidities. Stroke and co-morbidities are associated with raised levels of the pro-inflammatory cytokine interleukin-1 (IL-1). Inhibition of IL-1 by the administration of interleukin-1 receptor antagonist (IL-1Ra) has shown to be neuroprotective after experimental cerebral ischemia. Stroke can also trigger a robust neuroreparative response following injury, yet many of these new born neurons fail to survive or integrate into pre-existing circuits. Thus, we explore here effects of IL-1Ra on post-stroke neurogenesis in young and aged/co-morbid rats. Aged lean, aged Corpulent (a model of atherosclerosis, obesity and insulin resistance) and young Wistar male rats were exposed to transient cerebral ischemia, received subcutaneous IL-1Ra 3 and 6h during reperfusion, and effects on stroke outcome and neurogenesis were analyzed. Our results show that administration of IL-1Ra improves stroke outcome in both young and aged/co-morbid rats. Furthermore, IL-1Ra not only increases stem cell proliferation, but also significantly enhances neuroblast migration and the number of newly born neurons after cerebral ischemia. Overall, our data demonstrate that systemic administration of IL-1Ra improves outcome and promotes neurogenesis after experimental stroke, further highlighting the therapeutic potential of this clinically approved drug.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Neurogénesis/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Movimiento Celular/efectos de los fármacos , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Masculino , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The immune system is implicated in a wide range of disorders affecting the brain and is, therefore, an attractive target for therapy. Interleukin-1 (IL-1) is a potent regulator of the innate immune system important for host defense but is also associated with injury and disease in the brain. Here, we show that IL-1 is a key mediator driving an innate immune response to inflammatory challenge in the mouse brain but is dispensable in extracerebral tissues including the lung and peritoneum. We also demonstrate that IL-1α is an important ligand contributing to the CNS dependence on IL-1 and that IL-1 derived from the CNS compartment (most likely microglia) is the major source driving this effect. These data reveal previously unknown tissue-specific requirements for IL-1 in driving innate immunity and suggest that IL-1-mediated inflammation in the brain could be selectively targeted without compromising systemic innate immune responses that are important for resistance to infection. This property could be exploited to mitigate injury- and disease-associated inflammation in the brain without increasing susceptibility to systemic infection, an important complication in several neurological disorders.
Asunto(s)
Encéfalo/inmunología , Encefalitis/inmunología , Interleucina-1alfa/genética , Interleucina-1beta/genética , Microglía/inmunología , Transducción de Señal/inmunología , Animales , Encéfalo/patología , Encefalitis/inducido químicamente , Encefalitis/genética , Encefalitis/patología , Regulación de la Expresión Génica , Inmunidad Innata , Inyecciones Intraventriculares , Interleucina-1alfa/deficiencia , Interleucina-1alfa/inmunología , Interleucina-1beta/deficiencia , Interleucina-1beta/inmunología , Lipopolisacáridos , Pulmón/inmunología , Ratones , Ratones Noqueados , Microglía/patología , Infiltración Neutrófila , Neutrófilos/inmunología , Neutrófilos/patología , Especificidad de Órganos , Peritoneo/inmunologíaRESUMEN
Recent investigation of a constitutively active ADAMTS13 variant (caADAMTS13) in murine models of acute ischaemic stroke (AIS) have revealed a potential anti-inflammatory mechanism of action contributing to its protective effect. However, it remains unclear whether these observations are a direct result of VWF proteolysis by caADAMTS13. We have implemented state of the art in vitro assays of neutrophil rolling and transmigration to quantify the impact of caADAMTS13 on these processes. Moreover, we have tested caADAMTS13 in two in vivo assays of neutrophil migration to confirm the impact of the treatment on the neutrophil response to sterile inflammation. Neutrophil rolling, over an interleukin-1ß stimulated hCMEC/D3 monolayer, is directly inhibited by caADAMTS13, reducing the proportion of neutrophils rolling to 9.5 ± 3.8â¯% compared to 18.0 ± 4.5â¯% in untreated controls. Similarly, neutrophil transmigration recorded in real-time, was significantly suppressed in the presence of caADAMTS13 which reduced the number of migration events to a level like that in unstimulated controls (18.0 ± 4.5 and 15.8 ± 7.5 cells/mm2/h, respectively). Brain tissue from mice undergoing experimental focal cerebral ischaemia has indicated the inhibition of this process by caADAMTS13. This is supported by caADAMTS13's ability to reduce neutrophil migration into the peritoneal cavity in an ischaemia-independent model of sterile inflammation, with the VWF-dependent mechanism by which this occurs being confirmed using a second experimental stroke model. These findings will be an important consideration in the further development of caADAMTS13 as a potential therapy for AIS and other thromboinflammatory pathologies, including cardiovascular disease.
Asunto(s)
Proteína ADAMTS13 , Movimiento Celular , Modelos Animales de Enfermedad , Inflamación , Ratones Endogámicos C57BL , Neutrófilos , Trombosis , Animales , Neutrófilos/metabolismo , Neutrófilos/efectos de los fármacos , Trombosis/patología , Inflamación/patología , Inflamación/metabolismo , Ratones , Proteína ADAMTS13/metabolismo , Movimiento Celular/efectos de los fármacos , Humanos , Masculino , Rodamiento de Leucocito/efectos de los fármacos , Antiinflamatorios/farmacologíaRESUMEN
Interleukin-1α is a suggested dual-function cytokine that diverged from interleukin-1ß in mammals potentially by acquiring additional biological roles that relate to highly conserved regions in the pro-domain of interleukin-1α, including a nuclear localisation sequence and histone acetyltransferase-binding domains. Why evolution modified pro-interleukin-1α's subcellular location and protein interactome, and how this shaped interleukin-1α's intracellular role, is unknown. Here we show that TurboID proximity labelling with pro-interleukin-1α suggests a nuclear role for pro-interleukin-1α that involves interaction with histone acetyltransferases, including EP300. We also identify and validate inactivating mutations in the pro-interleukin-1α nuclear localisation sequence of multiple mammalian species, including toothed whales, castorimorpha and marsupials. However, histone acetyltransferase-binding domains are conserved in those species that have lost pro-interleukin-1α nuclear localisation. Together, these data suggest that histone acetyltransferase binding and nuclear localisation occurred together, and that while some species lost the nuclear localisation sequence in their pro-interleukin-1α, histone acetyltransferase binding ability was maintained. The nuclear localisation sequence was lost from several distinct species at different evolutionary times, suggesting convergent evolution, and that the loss of the nuclear localisation sequence confers some important biological outcome.
Asunto(s)
Núcleo Celular , Evolución Molecular , Interleucina-1alfa , Interleucina-1alfa/metabolismo , Interleucina-1alfa/genética , Animales , Núcleo Celular/metabolismo , Humanos , Proteína p300 Asociada a E1A/metabolismo , Proteína p300 Asociada a E1A/genética , Histona Acetiltransferasas/metabolismo , Histona Acetiltransferasas/genética , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Unión Proteica , Secuencia de AminoácidosRESUMEN
Blood-brain barrier (BBB) dysfunction occurs in many brain diseases, and there is increasing evidence to suggest that it is an early process in dementia which may be exacerbated by peripheral infection. Filter-exchange imaging (FEXI) is an MRI technique for measuring trans-membrane water exchange. FEXI data is typically analysed using the apparent exchange rate (AXR) model, yielding estimates of the AXR. Crusher gradients are commonly used to remove unwanted coherence pathways arising from longitudinal storage pulses during the mixing period. We first demonstrate that when using thin slices, as is needed for imaging the rodent brain, crusher gradients result in underestimation of the AXR. To address this, we propose an extended crusher-compensated exchange rate (CCXR) model to account for diffusion-weighting introduced by the crusher gradients, which is able to recover ground truth values of BBB water exchange (kin) in simulated data. When applied to the rat brain, kin estimates obtained using the CCXR model were 3.10 s-1 and 3.49 s-1 compared to AXR estimates of 1.24 s-1 and 0.49 s-1 for slice thicknesses of 4.0 mm and 2.5 mm respectively. We then validated our approach using a clinically relevant Streptococcus pneumoniae lung infection. We observed a significant 70 ± 10% increase in BBB water exchange in rats during active infection (kin = 3.78 ± 0.42 s-1) compared to before infection (kin = 2.72 ± 0.30 s-1; p = 0.02). The BBB water exchange rate during infection was associated with higher levels of plasma von Willebrand factor (VWF), a marker of acute vascular inflammation. We also observed 42% higher expression of perivascular aquaporin-4 (AQP4) in infected animals compared to non-infected controls, while levels of tight junction proteins remain consistent between groups. In summary, we propose a modelling approach for FEXI data which removes the bias in estimated water-exchange rates associated with the use of crusher gradients. Using this approach, we demonstrate the impact of peripheral infection on BBB water exchange, which appears to be mediated by endothelial dysfunction and associated with an increase in perivascular AQP4.
Asunto(s)
Barrera Hematoencefálica , Agua , Ratas , Animales , Barrera Hematoencefálica/metabolismo , Agua/metabolismo , Encéfalo/metabolismo , Imagen por Resonancia Magnética/métodos , Acuaporina 4/metabolismo , Pulmón/metabolismoRESUMEN
Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. Here, we used a combination of Drosophila and mouse, and, in the latter, both a pentylenetetrazole (PTZ)-induced seizure model and an electrically induced seizure model for refractory seizures to evaluate the anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode of action included increased expression of the firing rate homeostatic regulator Pumilio (PUM). Knockdown of pum expression, in Drosophila, blocked anticonvulsive effects of 4-TBB, while analysis of validated PUM targets in mouse brain revealed significant reductions following exposure to this compound. A structure-activity study identified the active parts of the molecule and, further, showed that the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-induced and electrically induced seizures. This study provides a proof of principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development.
Asunto(s)
Anticonvulsivantes , Pentilenotetrazol , Animales , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Benzaldehídos/efectos adversos , Drosophila , Homeostasis , Ratones , Neuronas , Pentilenotetrazol/efectos adversos , Pirazoles/uso terapéutico , Convulsiones/tratamiento farmacológico , Convulsiones/prevención & controlRESUMEN
Haemorrhagic stroke represents a significant public health burden, yet our knowledge and ability to treat this type of stroke are lacking. Previously we showed that we can target ischaemic-stroke lesions by selective translocation of lipid nanoparticles through the site of blood-brain barrier (BBB) disruption. The data we presented in this study provide compelling evidence that haemorrhagic stroke in mice induces BBB injury that mimics key features of the human pathology and, more importantly, provides a gate for entry of lipid nanoparticles-based therapeutics selectively to the bleeding site. Methods: Haemorrhagic stroke was induced in mice by intra-striatal collagenase injection. lipid nanoparticles were injected intravenously at 3 h, 24 h & 48 h post-haemorrhagic stroke and accumulation in the brain studied using in-vivo optical imaging and histology. BBB integrity, brain water content and iron accumulation were characterised using dynamic contrast-enhanced MRI, quantitative T1 mapping, and gradient echo MRI. Results: Using in-vivo SPECT/CT imaging and optical imaging revealed biphasic lipid nanoparticles entry into the bleeding site, with an early phase of increased uptake at 3-24 h post-haemorrhagic stroke, followed by a second phase at 48-72 h. Lipid nanoparticles entry into the brain post-haemorrhage showed an identical entry pattern to the trans-BBB leakage rate (Ktrans [min-1]) of Gd-DOTA, a biomarker for BBB disruption, measured using dynamic contrast-enhanced MRI. Discussion: Our findings suggest that selective accumulation of liposomes into the lesion site is linked to a biphasic pattern of BBB hyper-permeability. This approach provides a unique opportunity to selectively and efficiently deliver therapeutic molecules across the BBB, an approach that has not been utilised for haemorrhagic stroke therapy and is not achievable using free small drug molecules.
Asunto(s)
Accidente Cerebrovascular Hemorrágico , Accidente Cerebrovascular , Animales , Barrera Hematoencefálica/patología , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Humanos , Liposomas , Imagen por Resonancia Magnética/métodos , Ratones , Nanopartículas , Accidente Cerebrovascular/diagnóstico por imagen , Accidente Cerebrovascular/patologíaRESUMEN
Multi-diffusion-time diffusion-weighted MRI can probe tissue microstructure, but the method has not been widely applied to the microvasculature. At long diffusion-times, blood flow in capillaries is in the diffusive regime, and signal attenuation is dependent on blood velocity (v) and capillary segment length (l). It is described by the pseudo-diffusion coefficient (D*=vl/6) of intravoxel incoherent motion (IVIM). At shorter diffusion-times, blood flow is in the ballistic regime, and signal attenuation depends on v, and not l. In theory, l could be estimated using D* and v. In this study, we compare the accuracy and repeatability of three approaches to estimating v, and therefore l: the IVIM ballistic model, the velocity autocorrelation model, and the ballistic approximation to the velocity autocorrelation model. Twenty-nine rat datasets from two strains were acquired at 7 T, with b-values between 0 and 1000 smm-2 and diffusion times between 11.6 and 50 ms. Five rats were scanned twice to assess scan-rescan repeatability. Measurements of l were validated using corrosion casting and micro-CT imaging. The ballistic approximation of the velocity autocorrelation model had lowest bias relative to corrosion cast estimates of l, and had highest repeatability.
Asunto(s)
Velocidad del Flujo Sanguíneo/fisiología , Encéfalo/diagnóstico por imagen , Imagen de Difusión por Resonancia Magnética/métodos , Microvasos/fisiología , Animales , Encéfalo/irrigación sanguínea , Interpretación de Imagen Asistida por Computador , Modelos Biológicos , Ratas , Ratas Endogámicas F344 , Relación Señal-Ruido , Microtomografía por Rayos XRESUMEN
The development of effective therapies for stroke continues to face repeated translational failures. Brain endothelial cells form paracellular and transcellular barriers to many blood-borne therapies, and the development of efficient delivery strategies is highly warranted. Here, in a mouse model of stroke, we show selective recruitment of clinically used liposomes into the ischemic brain that correlates with biphasic blood brain barrier (BBB) breakdown. Intravenous administration of liposomes into mice exposed to transient middle cerebral artery occlusion took place at early (0.5 and 4 h) and delayed (24 and 48 h) time points, covering different phases of BBB disruption after stroke. Using a combination of in vivo real-time imaging and histological analysis we show that selective liposomal brain accumulation coincides with biphasic enhancement in transcellular transport followed by a delayed impairment to the paracellular barrier. This process precedes neurological damage in the acute phase and maintains long-term liposomal colocalization within the neurovascular unit, which could have great potential for neuroprotection. Levels of liposomal uptake by glial cells are similarly selectively enhanced in the ischemic region late after experimental stroke (2-3 days), highlighting their potential for blocking delayed inflammatory responses or shifting the polarization of microglia/macrophages toward brain repair. These findings demonstrate the capability of liposomes to maximize selective translocation into the brain after stroke and identify two windows for therapeutic manipulation. This emphasizes the benefits of selective drug delivery for efficient tailoring of stroke treatments.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Liposomas , Accidente Cerebrovascular/metabolismo , Animales , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Infarto de la Arteria Cerebral Media/metabolismo , Liposomas/química , Liposomas/farmacocinética , Liposomas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Transcitosis/efectos de los fármacosRESUMEN
Despite its long record of successful use in human vaccines, the mechanisms underlying the immunomodulatory effects of alum are not fully understood. Alum is a potent inducer of interleukin-1 (IL-1) secretion in vitro in dendritic cells and macrophages via Nucleotide-binding domain and leucine-rich repeat-containing (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome activation. However, the contribution of IL-1 to alum-induced innate and adaptive immune responses is controversial and the role of IL-1α following alum injection has not been addressed. This study shows that IL-1 is dispensable for alum-induced antibody and CD8 T cell responses to ovalbumin. However, IL-1 is essential for neutrophil infiltration into the injection site, while recruitment of inflammatory monocytes and eosinophils is IL-1 independent. Both IL-1α and IL-1ß are released at the site of injection and contribute to the neutrophil response. Surprisingly, these effects are NLRP3-inflammasome independent as is the infiltration of other cell populations. However, while NLRP3 and caspase 1 were dispensable, alum-induced IL-1ß at the injection site was dependent on the cysteine protease cathepsin S. Overall, these data demonstrate a previously unreported role for cathepsin S in IL-1ß secretion, show that inflammasome formation is dispensable for alum-induced innate immunity and reveal that IL-1α and IL-1ß are both necessary for alum-induced neutrophil influx in vivo.
Asunto(s)
Compuestos de Alumbre/farmacología , Inflamasomas , Interleucina-1alfa/inmunología , Interleucina-1beta/inmunología , Infiltración Neutrófila/efectos de los fármacos , Infiltración Neutrófila/inmunología , Vacunación , Compuestos de Alumbre/administración & dosificación , Animales , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
Circadian oscillators are autonomous molecular rhythms that reside in cells to align whole-organism physiology and behavior to the 24-h day. In flies, as in mammals, the oscillator operates in cells that coexpress CLOCK (CLK) and CYCLE (CYC). Recent work in Drosophila has shown that CLK is unique in its ability to generate heterologous oscillators, indicating that Clk gene expression defines the circadian cell fate. Here, using standard in vitro and in vivo techniques, we show that TWIN-OF-EYELESS (TOY; dPax6) regulates Clk expression in small ventrolateral neurons (s-LNvs) that coordinate sleep-wake cycles. Crucially, toy binds multiple sites at the Clk locus, is expressed independent of CLK-CYC in LNvs, regulates CLK protein levels under optimal photoperiodic conditions, and sets clock-speed during endogenous free-run. Furthermore, TOY is necessary for the onset of Clk expression in LNvs during embryogenesis. We propose that TOY contributes to a transcription complex that functions upstream of the oscillator to promote Clk expression in s-LNvs.
RESUMEN
The effect of recombinant human tissue plasminogen activator (rtPA) on neuroinflammation after stroke remains largely unknown. Here, we tested the effect of rtPA on expression of cellular adhesion molecules, chemokines, and cytokines, and compared those with levels of inflammatory cell recruitment, brain injury, and mortality over 3 days after transient middle cerebral artery occlusion (MCAO) in mice. Mortality was dramatically increased after rtPA treatment compared with saline treatment during the first day of reperfusion. Among the animals that survived, rtPA significantly increased CCL3 expression, microglia recruitment, and cerebral infarction 6 hours after MCAO. In contrast, the extent of neutrophils and macrophages infiltration in the brain was similar in both saline- and rtPA-treated animals. Recombinant human tissue plasminogen activator induced Il1b and Tnf expression, 6 and 72 hours after MCAO, respectively, and dramatically reduced interleukin 6 (IL-6) level 24 hours after reperfusion. A dose response study confirmed the effect of rtPA on CCL3 and Il1b expressions. The effect was similar at the doses of 1 and 10 mg/kg. In conclusion, we report for the first time that rtPA amplified microglia recruitment early after stroke in association with a rapid CCL3 production. This early response may take part in the higher susceptibility of rtPA-treated animals to reperfusion injury.
Asunto(s)
Quimiocina CCL3/inmunología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/inmunología , Activador de Tejido Plasminógeno/efectos adversos , Activador de Tejido Plasminógeno/inmunología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Encéfalo/patología , Quimiocina CCL3/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/patología , Inflamación/inducido químicamente , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Masculino , Ratones , Microglía/efectos de los fármacos , Microglía/inmunología , ARN Mensajero/genética , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Obesity is characterized by chronic inflammation associated with neutrophil and M1 macrophage infiltration into white adipose tissue. However, the mechanisms underlying this process remain largely unknown. Based on the ability of oil-based adjuvants to induce immune responses, we hypothesized that endogenous oils derived from necrotic adipocytes may function as an immunological "danger signal." Here we show that endogenous oils of human origin are potent adjuvants, enhancing antibody responses to a level comparable to Freund's incomplete adjuvant. The endogenous oils were capable of promoting interleukin (IL)-1α-dependent recruitment of neutrophils and M1-like macrophages, while simultaneously diminishing M2-like macrophages. We found that endogenous oils from subcutaneous and omental adipocytes, and from healthy and unhealthy obese individuals, promoted comparable inflammatory responses. Furthermore, we also confirmed that white adipocytes in visceral fat of metabolically unhealthy obese (MUO) individuals are significantly larger than those in metabolically healthy obese individuals. Since adipocyte size is positively correlated with adipocyte death, we propose that endogenous oils have a higher propensity to be released from hypertrophied visceral fat in MUO individuals and that this is the key factor in driving inflammation. In summary, this study shows that adipocytes contain a potent oil adjuvant which drives IL-1α-dependent proinflammatory responses in vivo.
Asunto(s)
Adipocitos , Tejido Adiposo/metabolismo , Inflamación/inmunología , Interleucina-1alfa/inmunología , Grasa Intraabdominal/metabolismo , Obesidad/inmunología , Aceites/farmacología , Animales , Femenino , Humanos , Inmunoterapia , Inflamación/tratamiento farmacológico , Mediadores de Inflamación/metabolismo , Resistencia a la Insulina , Masculino , Ratones , Obesidad/tratamiento farmacológicoRESUMEN
The CLOCK-CYCLE (CLK-CYC) heterodimer lies at the heart of the circadian oscillator mechanism in Drosophila, yet little is known about the identity of transcription factors that regulate the expression of Clk and/or cyc. Here, the authors have used a transgenic approach to isolate regions of the Clk locus that are necessary for expression in central oscillator neurons in the adult fly brain. This analysis shows that central clock cells can be subdivided into 2 distinct groups based on Clk gene regulation. Expression in the lateral neuron (LN), dorsal neuron 1 anterior (DN1a) and 2 (DN2) clusters requires cis-elements located in a 122 base-pair (bp) region (-206 to -84) of the Clk promoter. Expression in the remaining dorsal neurons, 1 posterior (DN1p) and 3 (DN3) and the lateral posterior neurons (LPN), requires regulatory elements located in the -856 to -206 region. In addition, expression in photoreceptors of the compound eye is enhanced by cis-elements located in a 3rd region of the Clk locus (-1982 to -856). This region also enhances expression in nonoscillator cells in the brain including the Kenyon cells, but expression in these neurons is suppressed by regulatory sites located further upstream of -1982. The authors' analysis reveals clear heterogeneity in Clk gene expression in the adult brain and provides a necessary focus to isolate novel transcription factors that bind at the Clk locus to regulate expression in different oscillator neuron subgroups. These results also suggest that the DN1a/DN2 neurons may have more molecular commonality with the LNs than they do with the DN1p/DN3/LPN neurons. Finally, this analysis has generated new transgenic lines that will enable genes to be misexpressed in subgroups of central oscillator cells that have previously been resistant to discrete genetic manipulation. Hence, these lines provide important new tools to facilitate a more complete dissection of the neural network that regulates output rhythms in physiology and behavior.
Asunto(s)
Proteínas CLOCK/genética , Proteínas de Drosophila/genética , Drosophila , Elementos de Facilitación Genéticos , Neuronas/fisiología , Regiones Promotoras Genéticas , Animales , Animales Modificados Genéticamente , Relojes Biológicos/fisiología , Proteínas CLOCK/metabolismo , Ritmo Circadiano/fisiología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Femenino , Regulación de la Expresión Génica , Masculino , Neuronas/citología , Fotoperiodo , TransgenesRESUMEN
Oxidative-stress resistance in Staphylococcus aureus is linked to metal ion homeostasis via several interacting regulators. In particular, PerR controls the expression of a regulon of genes, many of which encode antioxidants. Two PerR regulon members, ahpC (alkylhydroperoxide reductase) and katA (catalase), show compensatory regulation, with independent and linked functions. An ahpC mutation leads to increased H2O2 resistance due to greater katA expression via relief of PerR repression. Moreover, AhpC provides residual catalase activity present in a katA mutant. Mutation of both katA and ahpC leads to a severe growth defect under aerobic conditions in defined media (attributable to lack of catalase activity). This results in the inability to scavenge exogenous or endogenously produced H2O2, resulting in accumulation of H2O2 in the medium. This leads to DNA damage, the likely cause of the growth defect. Surprisingly, the katA ahpC mutant is not attenuated in two independent models of infection, which implies reduced oxygen availability during infection. In contrast, both AhpC and KatA are required for environmental persistence (desiccation) and nasal colonization. Thus, oxidative-stress resistance is an important factor in the ability of S. aureus to persist in the hospital environment and so contribute to the spread of human disease.