RESUMO
Microglia, the resident immune cells of the central nervous system (CNS), play a major role in damage progression and tissue remodeling after acute CNS injury, including ischemic stroke (IS) and spinal cord injury (SCI). Understanding the molecular mechanisms regulating microglial responses to injury may thus reveal novel therapeutic targets to promote CNS repair. Here, we investigated the role of microglial tumor necrosis factor receptor 2 (TNFR2), a transmembrane receptor previously associated with pro-survival and neuroprotective responses, in shaping the neuroinflammatory environment after CNS injury. By inducing experimental IS and SCI in Cx3cr1CreER:Tnfrsf1bfl/fl mice, selectively lacking TNFR2 in microglia, and corresponding Tnfrsf1bfl/fl littermate controls, we found that ablation of microglial TNFR2 significantly reduces lesion size and pro-inflammatory cytokine levels, and favors infiltration of leukocytes after injury. Interestingly, these effects were paralleled by opposite sex-specific modifications of microglial reactivity, which was found to be limited in female TNFR2-ablated mice compared to controls, whereas it was enhanced in males. In addition, we show that TNFR2 protein levels in the cerebrospinal fluid (CSF) of human subjects affected by IS and SCI, as well as healthy donors, significantly correlate with disease stage and severity, representing a valuable tool to monitor the inflammatory response after acute CNS injury. Hence, these results advance our understanding of the mechanisms regulating microglia reactivity after acute CNS injury, aiding the development of sex- and microglia-specific, personalized neuroregenerative strategies.
Assuntos
Microglia , Traumatismos da Medula Espinal , Animais , Feminino , Humanos , Masculino , Camundongos , Sistema Nervoso Central/metabolismo , Citocinas/metabolismo , Microglia/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Traumatismos da Medula Espinal/metabolismoRESUMO
The pleotropic cytokine tumor necrosis factor (TNF) is involved in the pathophysiology of multiple sclerosis (MS). In various models of MS, including experimental autoimmune encephalomyelitis (EAE), the membrane-bound form of TNF (tmTNF), which signals primarily via TNFR2, mediates protective and reparative effects, whereas the soluble form (solTNF), which signals primarily via TNFR1, promotes pro-inflammatory and detrimental functions. In this study, we investigated the role of TNFR2 expressed in oligodendrocytes in the early phase of EAE pathogenesis. We demonstrated that mice with specific ablation of oligodendroglial TNFR2 displayed early onset and higher peak of motor dysfunction when subjected to EAE, in advance of which accelerated infiltration of immune cells was observed as early as 10 days post EAE induction. The immune cell influx was preceded by microglial activation and increased blood brain barrier permeability. Lack of oligodendroglial TNFR2 accelerated the expression of inflammatory cytokines as well as expression and activation of the inflammasome. Gene expression profiling of oligodendrocytes sorted from the spinal cord 14 days post EAE induction showed robust upregulation of inflammatory genes, some of which were elevated in cells lacking TNFR2 compared to controls. Together, our data demonstrate that oligodendrocytes are directly involved in inflammation and immune modulation in CNS disease and this function is regulated, at least in part, by TNFR2.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Oligodendroglia/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Whether migraine headache and migraine aura share common pathophysiological mechanisms remains to be understood. Cilostazol causes cAMP accumulation and provokes migraine-like headache in migraine patients without aura. We investigated if cilostazol induces aura and migraine-like headache in patients with migraine with aura and alters peripheral endothelial function and levels of endothelial markers. In a randomized, double-blinded, placebo-controlled crossover study, 16 patients with migraine with aura (of whom 12 patients exclusively had attacks of migraine with aura) received 200 mg cilostazol (Pletal®) or placebo on two separate days. The development, duration, and characteristics of aura and headache were recorded using a questionnaire. Peripheral endothelial function was assessed by digital pulse amplitude tonometry using EndoPAT2000, and endothelial markers (VCAM1, E-selectin, and VEGFA) were measured. After administration of cilostazol, 14 patients (88%) experienced headache compared with six patients (38%) after placebo (P = 0.009). The headache in 12 patients (75%) after cilostazol and one patient (6%) after placebo fulfilled the criteria for migraine-like attacks (P = 0.0002). Patients reported that the attack mimicked the headache phase during their usual migraine attacks. However, aura symptoms were elicited in one patient after cilostazol and one patient after placebo. Further, endothelial function, as assessed by peripheral arterial tonometry, and endothelial markers were not significantly altered by cilostazol. Accumulation of cAMP by cilostazol induces migraine-like headache, but not aura, in patients with migraine with aura, even in those who exclusively reported attacks of migraine with aura in their spontaneous attacks. These findings further support dissociation between the aura and the headache phase with a yet unknown trigger for the aura and link between aura and headache. In addition, cilostazol administration did not significantly alter endothelial function, as assessed by peripheral arterial tonometry, or the endothelial markers, VCAM1, E-selectin, and VEGFA. However, post hoc analyses showed that our study was statistically underpowered for these outcomes.
Assuntos
Cilostazol/efeitos adversos , Transtornos de Enxaqueca/fisiopatologia , Enxaqueca com Aura/fisiopatologia , Vasodilatadores/efeitos adversos , Adulto , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos de Enxaqueca/induzido quimicamente , Enxaqueca com Aura/induzido quimicamenteRESUMO
The development of the rotator cuff enthesis is still poorly understood. The processes in the early and late developmental steps are gradually elucidated, but it is still unclear how cell activities are coordinated during development and maturation of the structured enthesis. This review summarizes current knowledge about development and age-related degradation of the supraspinatus enthesis. Healing and repair of an injured and degenerated supraspinatus enthesis also remain a challenge, as the original graded transitional tissue of the fibrocartilaginous insertion is not re-created after the tendon is surgically reattached to bone. Instead, mechanically inferior and disorganized tissue forms at the healing site because of scar tissue formation. Consequently, the enthesis never reaches mechanical properties comparable to those of the native enthesis. So far, no novel biologic healing approach has been successful in enhancing healing of the injured enthesis. The results revealed in this review imply the need for further research to pave the way for better treatment of patients with rotator cuff disorder.
Assuntos
Manguito Rotador/anatomia & histologia , Manguito Rotador/fisiologia , Animais , Fenômenos Biomecânicos/fisiologia , Citocinas/metabolismo , Expressão Gênica , Humanos , Inflamação/metabolismo , Metaloproteinases da Matriz/metabolismo , Lesões do Manguito Rotador/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , CicatrizaçãoRESUMO
Infiltration of myelin-specific T cells into the central nervous system induces the expression of proinflammatory cytokines in patients with multiple sclerosis (MS). We have previously shown that myelin-specific T cells are recruited into zones of axonal degeneration, where they stimulate lesion-reactive microglia. To gain mechanistic insight, we used RNA microarray analysis to compare the transcript profile in hippocampi from perforant pathway axonal-lesioned mice with and without adoptively transferred myelin-specific T cells 2 days postlesion, when microglia are clearly lesion reactive. Pathway analysis revealed that, among the 1,447 differently expressed transcripts, the interleukin (IL)-1 pathway including all IL-1 receptor ligands was upregulated in the presence of myelin-specific T cells. Quantitative polymerase chain reaction showed increased mRNA levels of IL-1ß, IL-1α, and IL-1 receptor antagonist in the T-cell-infiltrated hippocampi from axonal-lesioned mice. In situ hybridization and immunohistochemistry showed a T-cell-enhanced lesion-specific expression of IL-1ß mRNA and protein, respectively, and induction of the apoptosis-associated speck-like protein, ASC, in CD11b(+) cells. Double in situ hybridization showed colocalization of IL-1ß mRNA in a subset of CD11b mRNA(+) cells, of which many were part of cellular doublets or clusters, characteristic of proliferating, lesion-reactive microglia. Double-immunofluorescence showed a T-cell-enhanced colocalization of IL-1ß to CD11b(+) cells, including lesion-reactive CD11b(+) ramified microglia. These results suggest that myelin-specific T cells stimulate lesion-reactive microglial-like cells to produce IL-1ß. These findings are relevant to understand the consequences of T-cell infiltration in white and gray matter lesions in patients with MS.
Assuntos
Axônios/metabolismo , Interleucina-1beta/metabolismo , Microglia/patologia , Bainha de Mielina/patologia , Doenças Neurodegenerativas/patologia , Linfócitos T/fisiologia , Transferência Adotiva , Análise de Variância , Animais , Citocinas/genética , Citocinas/metabolismo , Giro Denteado/patologia , Modelos Animais de Doenças , Feminino , Fluoresceínas/metabolismo , Interleucina-1beta/genética , Camundongos , Análise em Microsséries , Infiltração de Neutrófilos , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/genéticaRESUMO
OBJECTIVE: Recent studies show that sublingual microcirculation is altered in patients resuscitated from CA. The objective of this study was to investigate whether the cerebral microcirculation is disturbed in the early post-resuscitation period. METHODS: Male Sprague-Dawley rats were randomized to either 10 minutes of CA or uninterrupted circulation, and observed to 120 or 360 minutes after ROSC. At 120 and 360 minutes, cerebral microcirculation was evaluated by SDF microscopy through a craniectomy. Plasma samples were analyzed for endothelial adhesion molecules and inflammatory markers, and brains were fixated for histological analysis. RESULTS: Cerebral microcirculation, evaluated by TVD, PVD, PPV, and MFI did not differ between groups (p > 0.16). Plasma samples drawn 360 minutes after ROSC displayed a significant increase in sE-selectin, sL-selectin, sI-CAM1, IL-1ß, IL-6, IL-10, and elastase compared to controls. In the CA animals, sE-selectin and elastase increased between 120 and 360 minutes after resuscitation (p < 0.007). Histological analysis revealed neuronal death in hippocampus layer CA1 360 min after resuscitation. CONCLUSION: When evaluated by SDF, the cerebral microcirculation appears unaffected in the early post-CA period despite hypotension, systemic inflammation, endothelial activation, and neuronal injury.
Assuntos
Encéfalo , Células Endoteliais , Parada Cardíaca , Mediadores da Inflamação/sangue , Microcirculação , Ressuscitação , Animais , Biomarcadores/sangue , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Parada Cardíaca/sangue , Parada Cardíaca/patologia , Parada Cardíaca/fisiopatologia , Parada Cardíaca/terapia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Interferon (IFN)-ß exerts anti-inflammatory effects, coupled to remarkable neurological improvements in multiple sclerosis, a neuroinflammatory condition of the central nervous system. Analogously, it has been hypothesized that IFN-ß, by limiting inflammation, decreases neuronal death and promotes functional recovery after stroke. However, the core actions of endogenous IFN-ß signaling in stroke are unclear. METHODS: To address this question, we used two clinically relevant models of focal cerebral ischemia, transient and permanent middle cerebral artery occlusion, and two genetically modified mouse lines, lacking either IFN-ß or its receptor, the IFN-α/ß receptor. Subsets of inflammatory and immune cells isolated from the brain, blood, and spleen were studied using flow cytometry. Sensorimotor deficits were assessed by a modified composite neuroscore, the rotating pole and grip strength tests, and cerebral infarct volumes were given by lack of neuronal nuclei immunoreactivity. RESULTS: Here, we report alterations in local and systemic inflammation in IFN-ß knockout (IFN-ßKO) mice over 8 days after induction of focal cerebral ischemia. Notably, IFN-ßKO mice showed a higher number of infiltrating leukocytes in the brain 2 days after stroke. Concomitantly, in the blood of IFN-ßKO mice, we found a higher percentage of total B cells but a similar percentage of mature and activated B cells, collectively indicating a higher proliferation rate. The additional differential regulation of circulating cytokines and splenic immune cell populations in wild-type and IFN-ßKO mice further supports an important immunoregulatory function of IFN-ß in stroke. Moreover, we observed a significant weight loss 2-3 days and a reduction in grip strength 2 days after stroke in the IFN-ßKO group, while endogenous IFN-ß signaling did not affect the infarct volume. CONCLUSIONS: We conclude that endogenous IFN-ß signaling attenuates local inflammation, regulates peripheral immune cells, and, thereby, may contribute positively to stroke outcome.
Assuntos
Isquemia Encefálica/patologia , Inflamação/patologia , Interferon beta , Acidente Vascular Cerebral/patologia , Animais , Linfócitos B/patologia , Encéfalo/patologia , Isquemia Encefálica/psicologia , Citocinas/sangue , Força da Mão/fisiologia , Infarto da Artéria Cerebral Média/patologia , Interferon beta/genética , Ataque Isquêmico Transitório/patologia , Leucócitos/patologia , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Equilíbrio Postural , Receptores de Interferon/genética , Baço/citologia , Baço/imunologia , Acidente Vascular Cerebral/psicologiaRESUMO
Beta-amyloid (Aß) plaques and chronic neuroinflammation are significant neuropathological features of Alzheimer's disease. Microglial cells in aged brains have potential to produce cytokines such as TNF and IL-1 family members (IL-1α, IL-1ß, and IL-1Ra) and to phagocytose Aß in Alzheimer's disease, however the inter-relationship between these processes is poorly understood. Here we show that % Aß plaque load followed a sigmoidal trajectory with age in the neocortex of APPswe/PS1ΔE9 Tg mice, and correlated positively with soluble Aß40 and Aß42. Aß measures were moderately correlated with mRNA levels of CD11b, TNF, and IL-1Ra. Cytokine production and Aß load were assessed in neocortical CD11b(+)(CD45(+)) microglia by flow cytometry. Whereas most microglia in aged mice produced IL-1Ra, relatively low proportions of microglia produced TNF, IL-1α, and IL-1ß. However, microglial production of these latter cytokines was generally increased in APP/PS1 Tg mice. Microglia that phagocytosed endogenously-produced Aß were only observed in APP/PS1 Tg mice. Differences in phagocytic index and total Aß load were observed in microglia with specific cytokine profiles. Both phagocytic index and total Aß load were higher in IL-1α(+) and IL-1Ra(+) microglia, than microglia that did not produce these cytokines. In contrast, total Aß load was lower in IL-1ß(+) and TNF(+) microglia, compared to IL-1ß(-) and TNF(-) microglia, and TNF(+) microglia also had a lower phagocytic index. Using GFP bone marrow chimeric mice, we confirmed that the majority of neocortical CD11b(+)(CD45(+)) microglia were resident cells (GFP(-)) in APP/PS1 Tg mice, even after selectively analysing CD11b(+)CD45(high) cells, which are typically considered to be infiltrating cells. Together, our data demonstrate that cytokine expression is selectively correlated with age and Aß pathology, and is associated with an altered Aß load in phagocytic microglia from APP/PS1 Tg mice. These findings have implications for understanding the regulation of microglial cytokine production and phagocytosis of Aß in Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Citocinas/metabolismo , Microglia/metabolismo , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Presenilina-1/genética , Presenilina-1/metabolismoRESUMO
Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)(2) (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.
Assuntos
Infarto da Artéria Cerebral Média/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Fármacos Neuroprotetores/uso terapêutico , Peptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Sítios de Ligação , Barreira Hematoencefálica , Cristalografia por Raios X , Proteína 4 Homóloga a Disks-Large , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Guanilato Quinases/antagonistas & inibidores , Humanos , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/patologia , Deficiências da Aprendizagem/etiologia , Deficiências da Aprendizagem/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Terapia de Alvo Molecular , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/prevenção & controle , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/farmacologia , Ressonância Magnética Nuclear Biomolecular , Domínios PDZ/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/farmacologia , Equilíbrio Postural , Conformação Proteica , Transtornos de Sensação/etiologia , Transtornos de Sensação/prevenção & controleRESUMO
BACKGROUND: Glial cell activation and overproduction of inflammatory mediators in the central nervous system (CNS) have been implicated in acute traumatic injuries to the CNS, including spinal cord injury (SCI). Elevated levels of the proinflammatory cytokine tumor necrosis factor (TNF), which exists in both a soluble (sol) and a transmembrane (tm) form, have been found in the lesioned cord early after injury. The contribution of solTNF versus tmTNF to the development of the lesion is, however, still unclear. METHODS: We tested the effect of systemically or centrally blocking solTNF alone, using XPro1595, versus using the drug etanercept to block both solTNF and tmTNF compared to a placebo vehicle following moderate SCI in mice. Functional outcomes were evaluated using the Basso Mouse Scale, rung walk test, and thermal hyperalgesia analysis. The inflammatory response in the lesioned cord was investigated using immunohistochemistry and western blotting analyses. RESULTS: We found that peripheral administration of anti-TNF therapies had no discernable effect on locomotor performances after SCI. In contrast, central administration of XPro1595 resulted in improved locomotor function, decreased anxiety-related behavior, and reduced damage to the lesioned spinal cord, whereas central administration of etanercept had no therapeutic effects. Improvements in XPro1595-treated mice were accompanied by increases in Toll-like receptor 4 and TNF receptor 2 (TNFR2) protein levels and changes in Iba1 protein expression in microglia/macrophages 7 and 28 days after SCI. CONCLUSIONS: These studies suggest that, by selectively blocking solTNF, XPro1595 is neuroprotective when applied directly to the lesioned cord. This protection may be mediated via alteration of the inflammatory environment without suppression of the neuroprotective effects of tmTNF signaling through TNFR2.
Assuntos
Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Feminino , Bombas de Infusão Implantáveis , Injeções Epidurais , Injeções Subcutâneas , Camundongos , Traumatismos da Medula Espinal/metabolismo , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Crosstalk between the immune system in the brain and the periphery may contribute to the long-term outcome both in experimental and clinical stroke. Although, the immune defense collectin surfactant protein-D (SP-D) is best known for its role in pulmonary innate immunity, SP-D is also known to be involved in extrapulmonary modulation of inflammation in mice. We investigated whether SP-D affected cerebral ischemic infarction and ischemia-induced inflammatory responses in mice. METHODS: The effect of SP-D was studied by comparing the size of ischemic infarction and the inflammatory and astroglial responses in SP-D knock out (KO) and wild type (WT) mice subjected to permanent middle cerebral artery occlusion. SP-D mRNA production was assessed in isolated cerebral arteries and in the whole brain by PCR, and SP-D protein in normal appearing and ischemic human brain by immunohistochemistry. Changes in plasma SP-D and TNF were assessed by ELISA and proximity ligation assay, respectively. RESULTS: Infarct volumetric analysis showed that ablation of SP-D had no effect on ischemic infarction one and five days after induction of ischemia. Further, ablation of SP-D had no effect on the ischemia-induced increase in TNF mRNA production one day after induction of ischemia; however the TNF response to the ischemic insult was affected at five days. SP-D mRNA was not detected in parenchymal brain cells in either naïve mice or in mice subjected to focal cerebral ischemia. However, SP-D mRNA was detected in middle cerebral artery cells in WT mice and SP-D protein in vascular cells both in normal appearing and ischemic human brain tissue. Measurements of the levels of SP-D and TNF in plasma in mice suggested that levels were unaffected by the ischemic insult. Microglial-leukocyte and astroglial responses were comparable in SP-D KO and WT mice. CONCLUSIONS: SP-D synthesis in middle cerebral artery cells is consistent with SP-D conceivably leaking into the infarcted area and affecting local cytokine production. However, there was no SP-D synthesis in parenchymal brain cells and ablation of SP-D had no effect on ischemic cerebral infarction.
Assuntos
Infarto Cerebral/metabolismo , Proteína D Associada a Surfactante Pulmonar/sangue , Proteína D Associada a Surfactante Pulmonar/deficiência , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Antígeno CD11b/metabolismo , Artérias Cerebrais/metabolismo , Artérias Cerebrais/patologia , Infarto Cerebral/etiologia , Infarto Cerebral/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Infarto da Artéria Cerebral Média/complicações , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteína D Associada a Surfactante Pulmonar/genética , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Astrocytes are taking the center stage in neurotrauma and neurological diseases as they appear to play a dominant role in the inflammatory processes associated with these conditions. Previously, we reported that inhibiting NF-κB activation in astrocytes, using a transgenic mouse model (GFAP-IκBα-dn mice), results in improved functional recovery, increased white matter preservation and axonal sparing following spinal cord injury (SCI). In the present study, we sought to determine whether this improvement, due to inhibiting NF-κB activation in astrocytes, could be the result of enhanced oligodendrogenesis in our transgenic mice. METHODS: To assess oligodendrogenesis in GFAP-IκBα-dn compared to wild-type (WT) littermate mice following SCI, we used bromodeoxyuridine labeling along with cell-specific immuno-histochemistry, confocal microscopy and quantitative cell counts. To further gain insight into the underlying molecular mechanisms leading to increased white matter, we performed a microarray analysis in naïve and 3 days, 3 and 6 weeks following SCI in GFAP-IκBα-dn and WT littermate mice. RESULTS: Inhibition of astroglial NF-κB in GFAP-IκBα-dn mice resulted in enhanced oligodendrogenesis 6 weeks following SCI and was associated with increased levels of myelin proteolipid protein compared to spinal cord injured WT mice. The microarray data showed a large number of differentially expressed genes involved in inflammatory and immune response between WT and transgenic mice. We did not find any difference in the number of microglia/leukocytes infiltrating the spinal cord but did find differences in their level of expression of toll-like receptor 4. We also found increased expression of the chemokine receptor CXCR4 on oligodendrocyte progenitor cells and mature oligodendrocytes in the transgenic mice. Finally TNF receptor 2 levels were significantly higher in the transgenic mice compared to WT following injury. CONCLUSIONS: These studies suggest that one of the beneficial roles of blocking NF-κB in astrocytes is to promote oligodendrogenesis through alteration of the inflammatory environment.
Assuntos
Astrócitos/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/fisiologia , Neurogênese/fisiologia , Oligodendroglia/fisiologia , Traumatismos da Medula Espinal/metabolismo , Animais , Astrócitos/patologia , Feminino , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Transgênicos , Oligodendroglia/patologia , Traumatismos da Medula Espinal/patologia , Regulação para Cima/fisiologiaRESUMO
Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major contributor to physiological and pathological glutamate-mediated Ca2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented γ-hydroxybutyrate (GHB) ligands as the first small molecules selectively targeting and stabilizing the CaMKIIα hub domain. Here, we report that the cyclic GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), improves sensorimotor function after experimental stroke in mice when administered at a clinically relevant time and in combination with alteplase. Further, we observed improved hippocampal neuronal activity and working memory after stroke. On the biochemical level, we observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated ischemia-specific expression of a constitutively active CaMKII kinase proteolytic fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. Similarly, HOCPCA's effects on dampening inflammatory changes require further investigation as an underlying protective mechanism. HOCPCA's selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKIIα hub domain as an attractive neuroprotective strategy.
Assuntos
Oxibato de Sódio , Acidente Vascular Cerebral , Camundongos , Animais , Oxibato de Sódio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , CogniçãoRESUMO
Background and aims: Glucagon-like peptide 1 (GLP-1) receptor agonists (GLP-1RA) are widely used for the treatment of type 2 diabetes, and recent studies indicate that they may be cardio- and neuroprotective. The safety and effect of a single dose of exenatide, a short-acting GLP-1RA, on cerebral and peripheral arterial function remain unknown. Methods: In this randomized, double-blind pilot trial, we assigned elderly healthy volunteers without diabetes and no previous history of stroke to receive a single dose of subcutaneous exenatide (5 µg) or placebo. Primary outcome was immediate changes over time in blood flow velocity of the middle cerebral arteries (VMCA) assessed by repeated transcranial Doppler measurements. Secondary outcomes were changes in peripheral arterial function with finger plethysmography, ankle-brachial index (ABI), and inflammatory- and endothelial-specific biomarkers. Results: Healthy volunteers (13 women and 17 men) were included: (mean ± standard deviation) age: 62 ± 8 years; body weight: 79.6 ± 12.7 kg; VMCA: 65.3 ± 10.7 cm/s; fasting plasma glucose: 5.5 ± 0.5 mmol/L; HbA1c: 33.9 ± 4.1 mmol/mol (5.3 ± 0.38%). No differences between exenatide and placebo group were seen regarding VMCA (p = 0.058), systolic ABI (p = 0.71), plethysmography (p = 0.45), tumor necrosis factor (p = 0.33), interleukin-6 (p = 0.11), interleukin-1ß (p = 0.34), vascular cell adhesion molecule 1 (p = 0.73), intercellular adhesion molecule 1 (p = 0.74), or E-selectin (p = 0.31). No severe adverse events were observed. Conclusion: A single dose of exenatide did not change cerebral blood flow velocity or peripheral vessel function in elderly healthy volunteers. The medication was safe to use in persons without diabetes allowing us to investigate this drug further in search of the neuroprotective mechanisms. Clinical Trial Registration: https://clinicaltrials.gov, Identifier NCT02838589.
RESUMO
METHODS: Cerebrospinal fluid (CSF) and plasma levels of 38 biomarkers from 20 neurosarcoidosis (NS) patients were compared to healthy controls (HC). RESULTS: In CSF, 25 biomarkers were significantly elevated compared to HC: IFNγ, TNFα, TNFß, IL-2, IL-6, IL-10, IL-12B, IL-15, IL-16, CCL2, CCL3, CCL4, CCL11, CCL13, CCL17, CCL22, CCL26, CXCL8, CXCL10, TNFR2, VEGF-A, PIGF, SAA, VCAM1, and ICAM1. In plasma, 12 biomarkers were significantly elevated compared to HC: IFNγ, TNFα, CCL2, CCL3, CCL4, CCL17, CXCL10, VEGFR1, PIGF, SAA, VCAM1, and ICAM1. CONCLUSION: NS patients have profoundly elevated cytokines, chemokines, vascular angiogenesis, and vascular injury biomarkers in CSF and plasma.
Assuntos
Doenças do Sistema Nervoso Central , Quimiocinas , Citocinas , Sarcoidose , Biomarcadores , Doenças do Sistema Nervoso Central/sangue , Doenças do Sistema Nervoso Central/líquido cefalorraquidiano , Humanos , Sarcoidose/sangue , Sarcoidose/líquido cefalorraquidianoRESUMO
METHODS: Twenty patients with newly diagnosed neurosarcoidosis were examined for multiple outcomes in an observational cohort study with 12-month follow-up. RESULTS: The patients' contrast-enhancing lesions on MRI scans reduced during treatment (p < 0.0001). The mean modified Rankin Score improved from 3.0 to 1.8 (p < 0.0001), and 75% of patients experienced clinically important improvement. Patients improved on the Symbol Digit Modalities Test (p < 0.0001) and on SF-36 Physical (p = 0.003) and Mental Component Summary scores (p = 0.03). Proportions of patients with substantial fatigue (75%) and high depression score (35%) were unchanged. CONCLUSIONS: 12-month immunosuppression improved several outcomes, and 75% of patients experienced clinically important improvement.
Assuntos
Doenças do Sistema Nervoso Central , Doenças do Sistema Nervoso Central/diagnóstico por imagem , Seguimentos , Humanos , Testes Neuropsicológicos , Estudos Prospectivos , SarcoidoseRESUMO
Increasing evidence demonstrates that inflammatory cytokines-such as tumor necrosis factor (TNF)-are produced at low levels in the brain under physiological conditions and may be crucial for synaptic plasticity, neurogenesis, learning and memory. Here, we examined the effects of developmental TNF deletion on spatial learning and memory using 11-13-month-old TNF knockout (KO) and C57BL6/J wild-type (WT) mice. The animals were tested in the Barnes maze (BM) arena under baseline conditions and 48 h following an injection of the endotoxin lipopolysaccharide (LPS), which was administered at a dose of 0.5 mg/kg. Vehicle-treated KO mice were impaired compared to WT mice during the acquisition and memory-probing phases of the BM test. No behavioral differences were observed between WT and TNF-KO mice after LPS treatment. Moreover, there were no differences in the hippocampal content of glutamate and noradrenaline between groups. The effects of TNF deletion on spatial learning and memory were observed in male, but not female mice, which were not different compared to WT mice under baseline conditions. These results indicate that TNF is required for spatial learning and memory in male mice under physiological, non-inflammatory conditions, however not following the administration of LPS. Inflammatory signalling can thereby modulate spatial cognition in male subjects, highlighting the importance of sex- and probably age-stratified analysis when examining the role of TNF in the brain.
Assuntos
Imunidade , Memória , Aprendizagem Espacial , Fator de Necrose Tumoral alfa/metabolismo , Animais , Cognição/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Hipocampo/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurotransmissores/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
OBJECTIVE: Aberrant pyrin inflammasome activity triggers familial Mediterranean fever (FMF) pathogenesis, but the exact mechanism remains elusive and an obstacle to efficient treatment. We undertook this study to identify pyrin inflammasome-specific mechanisms to improve FMF treatment and diagnostics in the future. METHODS: Pyrin-specific protein secretion was assessed by proteome analysis in U937-derived macrophages, and specific findings were confirmed in pyrin inflammasome-activated monocytes from healthy blood donors and patients with FMF, stratified according to MEFV genotype categories corresponding to a suspected increase in FMF disease severity. RESULTS: Proteome data revealed a differential secretion pattern of interleukin-1 receptor antagonist (IL-1Ra) from pyrin- and NLRP3-activated U937-derived macrophages, which was verified by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Moreover, pyrin activation significantly reduced IL1RN messenger RNA expression (P < 0.001) and IL-1Ra secretion (P < 0.01) in healthy donor and FMF monocytes, respectively. Independent of MEFV genotype, unstimulated FMF monocytes from colchicine-treated patients secreted lower amounts of IL-1Ra compared to healthy donors (P < 0.05) and displayed decreased ratios of IL-1Ra:IL-1ß (P < 0.05), suggesting a reduced antiinflammatory capacity. CONCLUSION: Our data show an inherent lack of IL-1Ra expression specific to pyrin inflammasome activation, suggesting a new mechanism underlying FMF pathogenesis. The reduced IL-1Ra levels in FMF monocytes suggest a diminished antiinflammatory capacity that potentially leaves FMF patients sensitive to proinflammatory stimuli, regardless of receiving colchicine therapy. Thus, considering the potential clinical consequence of reduced monocyte IL-1Ra secretion in FMF patients, we suggest further investigation into IL-1Ra dynamics and its potential implications for FMF treatment in the future.
Assuntos
Febre Familiar do Mediterrâneo/metabolismo , Inflamassomos/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Pirina/metabolismo , Linhagem Celular , Febre Familiar do Mediterrâneo/genética , Humanos , Proteína Antagonista do Receptor de Interleucina 1/genética , Macrófagos/metabolismo , Monócitos/metabolismo , Proteoma , Pirina/genéticaRESUMO
Tumor necrosis factor receptor 1 and 2 (TNFR1 and TNFR2) have been found in brain parenchyma of stroke patients, and plasma levels are increased in the acute phase of stroke. We evaluated associations between TNFR1 and TNFR2 plasma levels and stroke severity, infarct size, and functional outcome. Furthermore, we examined cellular expression of TNFR1 and TNFR2 on leukocyte subpopulations to explore the origin of the increased receptor levels. Blood samples were taken from 33 acute ischemic stroke patients and 10 healthy controls. TNFR1 and TNFR2 plasma concentrations were measured and correlated against the Scandinavian Stroke Scale at admission, infarct volume, and the modified Rankin Scale score three months after stroke onset. Classical, intermediate, and non-classical monocytes as well as neutrophils were purified, and cellular expression of TNFR1 and TNFR2 was examined using flow cytometry. TNFR1 and TNFR2 plasma levels were both increased after ischemic stroke, but we found no correlation with patient outcome measurements. Compared to healthy controls, ischemic stroke patients had decreased non-classical monocyte and neutrophil populations expressing TNFR1 and increased neutrophils expressing TNFR2, and decreased non-classical populations co-expressing both TNFR1 and TNFR2. This study supports the hypothesis of an acute immunological response orchestrated by the peripheral immune system following an ischemic stroke. However, the origin of the increased TNFR1 and TNFR2 plasma levels could not be clearly linked to peripheral monocytes or neutrophils. Future studies are needed and will help clarify the potential role as treatment target.