RESUMO
AIMS: The objective of the study is to explore the importance of tissue hypoxia in causing neurological deficits and demyelination in the inflamed CNS, and the value of inspiratory oxygen treatment, using both active and passive experimental autoimmune encephalomyelitis (EAE). METHODS: Normobaric oxygen treatment was administered to Dark Agouti rats with either active or passive EAE, compared with room air-treated, and naïve, controls. RESULTS: Severe neurological deficits in active EAE were significantly improved after just 1 h of breathing approximately 95% oxygen. The improvement was greater and more persistent when oxygen was applied either prophylactically (from immunisation for 23 days), or therapeutically from the onset of neurological deficits for 24, 48, or 72 h. Therapeutic oxygen for 72 h significantly reduced demyelination and the integrated stress response in oligodendrocytes at the peak of disease, and protected from oligodendrocyte loss, without evidence of increased oxidative damage. T-cell infiltration and cytokine expression in the spinal cord remained similar to that in untreated animals. The severe neurological deficit of animals with passive EAE occurred in conjunction with spinal hypoxia and was significantly reduced by oxygen treatment initiated before their onset. CONCLUSIONS: Severe neurological deficits in both active and passive EAE can be caused by hypoxia and reduced by oxygen treatment. Oxygen treatment also reduces demyelination in active EAE, despite the autoimmune origin of the disease.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Ratos , Animais , Camundongos , Esclerose Múltipla/metabolismo , Medula Espinal/metabolismo , Hipóxia/metabolismo , Oxigênio/metabolismo , Oxigênio/uso terapêutico , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: Treatment of relapses in multiple sclerosis (MS) has not advanced beyond steroid use, which reduces acute loss of function, but has little effect on residual disability. Acute loss of function in an MS model (experimental autoimmune encephalomyelitis [EAE]) is partly due to central nervous system (CNS) hypoxia, and function can promptly improve upon breathing oxygen. Here, we investigate the cause of the hypoxia and whether it is due to a deficit in oxygen supply arising from impaired vascular perfusion. We also explore whether the CNS-selective vasodilating agent, nimodipine, may provide a therapy to restore function, and protect from demyelination in 2 MS models. METHODS: A variety of methods have been used to measure basic cardiovascular physiology, spinal oxygenation, mitochondrial function, and tissue perfusion in EAE. RESULTS: We report that the tissue hypoxia in EAE is associated with a profound hypoperfusion of the inflamed spinal cord. Treatment with nimodipine restores spinal oxygenation and can rapidly improve function. Nimodipine therapy also reduces demyelination in both EAE and a model of the early MS lesion. INTERPRETATION: Loss of function in EAE, and demyelination in EAE, and the model of the early MS lesion, seem to be due, at least in part, to tissue hypoxia due to local spinal hypoperfusion. Therapy to improve blood flow not only protects neurological function but also reduces demyelination. We conclude that nimodipine could be repurposed to offer substantial clinical benefit in MS. ANN NEUROL 2020 ANN NEUROL 2020;88:123-136.
Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Nimodipina/uso terapêutico , Medula Espinal/patologia , Animais , Progressão da Doença , Encefalomielite Autoimune Experimental/patologia , Feminino , Imageamento por Ressonância Magnética , Masculino , Bainha de Mielina/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Hyperspectral imaging (HSI) systems have the potential to retrieve in vivo hemodynamic and metabolic signals from the exposed cerebral cortex. The use of multiple narrow wavelength bands in the near infrared (NIR) range theoretically allows not only to image brain tissue oxygenation and hemodynamics via mapping of hemoglobin concentration changes, but also to directly quantify cerebral metabolism via measurement of the redox states of mitochondrial cytochrome-c-oxidase (CCO). The aim of this study is to assess the possibility of performing hyperspectral imaging of in vivo cerebral oxyhemoglobin (HbO2), deoxyhemoglobin (HHb) and oxidized CCO (oxCCO) using commercially available HSI devices. For this reason, a hyperspectral snapshot solution based on Cubert GmbH technology (S185 FireflEYE camera) has been tested on the exposed cortex of mice during normoxic, hypoxic and hyperoxic conditions. The system allows simultaneous acquisition of 138 wavelength bands between 450 and 998 nm, with spectral sampling and resolution of ~4 to 8 nm. From the hyperspectral data, relative changes in concentration of hemoglobin and oxCCO are estimated and hemodynamic and metabolic maps of the imaged cortex are calculated for two different NIR spectral ranges. Spectroscopic analysis at particular regions of interest is also performed, showing typical oxygen-dependent hemodynamic responses. The results highlight some of the potentials of the technology, but also the limitations of the tested commercial solution for such specific application, in particular regarding spatial resolution.
Assuntos
Mapeamento Encefálico/métodos , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , Imagem Óptica/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Complexo IV da Cadeia de Transporte de Elétrons/análise , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hemodinâmica/fisiologia , Hemoglobinas/análise , Hemoglobinas/metabolismo , CamundongosRESUMO
OBJECTIVE: Demyelination is a cardinal feature of multiple sclerosis, but it remains unclear why new lesions form, and whether they can be prevented. Neuropathological evidence suggests that demyelination can occur in the relative absence of lymphocytes, and with distinctive characteristics suggestive of a tissue energy deficit. The objective was to examine an experimental model of the early multiple sclerosis lesion and identify pathogenic mechanisms and opportunities for therapy. METHODS: Demyelinating lesions were induced in the rat spinal dorsal column by microinjection of lipopolysaccharide, and examined immunohistochemically at different stages of development. The efficacy of treatment with inspired oxygen for 2 days following lesion induction was evaluated. RESULTS: Demyelinating lesions were not centered on the injection site, but rather formed 1 week later at the white-gray matter border, preferentially including the ventral dorsal column watershed. Lesion formation was preceded by a transient early period of hypoxia and increased production of superoxide and nitric oxide. Oligodendrocyte numbers decreased at the site shortly afterward, prior to demyelination. Lesions formed at a site of inherent susceptibility to hypoxia, as revealed by exposure of naive animals to a hypoxic environment. Notably, raising the inspired oxygen (80%, normobaric) during the hypoxic period significantly reduced or prevented the demyelination. INTERPRETATION: Demyelination characteristic of at least some early multiple sclerosis lesions can arise at a vascular watershed following activation of innate immune mechanisms that provoke hypoxia, and superoxide and nitric oxide formation, all of which can compromise cellular energy sufficiency. Demyelination can be reduced or eliminated by increasing inspired oxygen to alleviate the transient hypoxia.
Assuntos
Doenças Desmielinizantes , Hipóxia , Oxigenoterapia , Medula Espinal , Animais , Doenças Desmielinizantes/imunologia , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/prevenção & controle , Modelos Animais de Doenças , Hipóxia/imunologia , Hipóxia/metabolismo , Hipóxia/patologia , Hipóxia/prevenção & controle , Lipopolissacarídeos , Masculino , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Esclerose Múltipla/prevenção & controle , Ratos , Ratos Sprague-Dawley , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
OBJECTIVE: To explore the presence and consequences of tissue hypoxia in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). METHODS: EAE was induced in Dark Agouti rats by immunization with recombinant myelin oligodendrocyte glycoprotein and adjuvant. Tissue hypoxia was assessed in vivo using 2 independent methods: an immunohistochemical probe administered intravenously, and insertion of a physical, oxygen-sensitive probe into the spinal cord. Indirect markers of tissue hypoxia (eg, expression of hypoxia-inducible factor-1α [HIF-1α], vessel diameter, and number of vessels) were also assessed. The effects of brief (1 hour) and continued (7 days) normobaric oxygen treatment on function were evaluated in conjunction with other treatments, namely administration of a mitochondrially targeted antioxidant (MitoQ) and inhibition of inducible nitric oxide synthase (1400W). RESULTS: Observed neurological deficits were quantitatively, temporally, and spatially correlated with spinal white and gray matter hypoxia. The tissue expression of HIF-1α also correlated with loss of function. Spinal microvessels became enlarged during the hypoxic period, and their number increased at relapse. Notably, oxygen administration significantly restored function within 1 hour, with improvement persisting at least 1 week with continuous oxygen treatment. MitoQ and 1400W also caused a small but significant improvement. INTERPRETATION: We present chemical, physical, immunohistochemical, and therapeutic evidence that functional deficits caused by neuroinflammation can arise from tissue hypoxia, consistent with an energy crisis in inflamed central nervous system tissue. The neurological deficit was closely correlated with spinal white and gray matter hypoxia. This realization may indicate new avenues for therapy of neuroinflammatory diseases such as MS.
Assuntos
Encefalomielite Autoimune Experimental/fisiopatologia , Hipóxia/fisiopatologia , Inflamação/fisiopatologia , Oxigênio/farmacologia , Doenças da Medula Espinal/fisiopatologia , Amidinas/farmacologia , Animais , Benzilaminas/farmacologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Hipóxia/induzido quimicamente , Hipóxia/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Micronutrientes/farmacologia , Compostos Organofosforados/farmacologia , Oxigênio/administração & dosagem , Ratos , Recuperação de Função Fisiológica/efeitos dos fármacos , Índice de Gravidade de Doença , Método Simples-Cego , Doenças da Medula Espinal/induzido quimicamente , Doenças da Medula Espinal/tratamento farmacológico , Ubiquinona/análogos & derivados , Ubiquinona/farmacologiaRESUMO
OBJECTIVE: To determine the serum cytokine profiles of patients with spinal cord injury (SCI) and varying clinical presentations relative to healthy, able-bodied, age-matched control subjects. DESIGN: Cross-sectional study. SETTING: Clinical research unit. PARTICIPANTS: People with SCI (N=56) and different clinical presentations, and healthy, able-bodied, age-matched control subjects (N=35). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Serum levels of the proinflammatory cytokines interleukin (IL) 1beta, IL-6, tumor necrosis factor alpha (TNF-alpha), the anti-inflammatory cytokines IL-4 and IL-10, the regulatory cytokine IL-2, the IL-1 receptor antagonist (IL-1RA), and autoantibodies against myelin-associated glycoprotein and GM(1) ganglioside (anti-GM(1)) immunoglobulin (IgG and IgM), as determined by enzyme-linked immunosorbent assay. The relationship between elevated serum cytokine levels and clinical variables was also studied. RESULTS: SCI subjects exhibited serum concentrations of IL-6, TNF-alpha, IL-1RA, and anti-GM(1) (IgG) that were greater (P<.05) than control group values. Elevated cytokine concentrations were not associated with high white blood cell counts, level of injury, or American Spinal Injury Association classification; they were evident in SCI subjects who were asymptomatic for medical complications, but were further elevated in subjects with pain, urinary tract infection (UTI), and pressure ulcers. CONCLUSIONS: Elevated levels of circulating proinflammatory cytokines and autoantibodies are present in the serum of SCI subjects without medical complications, and are further elevated in SCI subjects with neuropathic pain, UTI, or pressure ulcers, relative to healthy, able-bodied control subjects. These findings may be indicative of a protective autoimmunity, simply a consequence of occult or evident infection, or evidence of cytokine dysregulation that may contribute to an immune-mediated impairment of axonal conduction.
Assuntos
Autoanticorpos/sangue , Citocinas/sangue , Traumatismos da Medula Espinal/imunologia , Adulto , Vértebras Cervicais/lesões , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Gangliosídeo G(M1)/imunologia , Humanos , Proteína Antagonista do Receptor de Interleucina 1/sangue , Interleucina-8/sangue , Masculino , Glicoproteína Associada a Mielina/imunologia , Paraplegia/imunologia , Úlcera por Pressão/imunologia , Quadriplegia/imunologia , Valores de Referência , Vértebras Torácicas/lesões , Fator de Necrose Tumoral alfa/sangue , Infecções Urinárias/imunologiaRESUMO
Quantification of blood oxygen saturation (SO2) in vivo is essential for understanding the pathogenesis of diseases in which hypoxia is thought to play a role, including inflammatory disorders such as multiple sclerosis (MS) and rheumatoid arthritis (RA). We describe a low-cost multispectral microscope and oximetry technique for calibration-free absolute oximetry of surgically exposed blood vessels in vivo. We imaged the vasculature of the dorsal spinal cord in healthy rats, and varied inspired oxygen (FiO2) in order to evaluate the sensitivity of the imaging system to changes in SO2. The venous SO2 was calculated as 67.8 ± 10.4% (average ± standard deviation), increasing to 83.1 ± 11.6% under hyperoxic conditions (100% FiO2) and returning to 67.4 ± 10.9% for a second normoxic period; the venous SO2 was 50.9 ± 15.5% and 29.2 ± 24.6% during subsequent hypoxic states (18% and 15% FiO2 respectively). We discuss the design and performance of our multispectral imaging system, and the future scope for extending this oximetry technique to quantification of hypoxia in inflamed tissue.
Assuntos
Microscopia , Oximetria/métodos , Medula Espinal/irrigação sanguínea , Medula Espinal/diagnóstico por imagem , Algoritmos , Animais , Feminino , Processamento de Imagem Assistida por Computador , Inalação , Oxigênio/sangue , Ratos , Veias/fisiologiaRESUMO
Increased expression of the proinflammatory cytokine tumor necrosis factor-alpha (TNFalpha) and its soluble receptors is evident within the central nervous system (CNS) following traumatic brain injury and spinal cord injury. TNFalpha is integral to the acute inflammatory cascade that follows neurotrauma and has been shown to have both beneficial and detrimental properties. We examined the effects of varying concentrations (1-5000 ng/mL) of recombinant human TNFalpha (rhTNFalpha) on select electrophysiological properties of excised guinea pig spinal cord tissue. Pulsed electrical stimuli (0.33 Hz) were delivered to strips of isolated ventral white matter in a double sucrose gap chamber. Recordings were made of the compound action potential (CAP) and membrane potential before, during, and after bathing the tissue with rhTNFalpha for 30 min. Increasing concentrations of rhTNFalpha yielded progressively greater reductions in amplitude of the CAP that were temporally associated with depolarization of the resting compound membrane potential. These effects were largely reversed on washout of rhTNFalpha and were not present when heat-denatured rhTNFalpha was introduced. The results provide evidence that elevated concentrations of TNFalpha induce reversible depolarization of the compound membrane potential and reduction in CAP amplitude, sometimes to the point of extinction of the CAP, suggestive of impaired axonal conduction. These observations point to a new mechanism of immune-mediated central conduction deficit. Cytokine-induced alterations in membrane properties and axonal conduction may contribute to neurological deficits following CNS injury by compounding trauma-induced myelinopathy and axonopathy.
Assuntos
Axônios/fisiologia , Medula Espinal/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletrofisiologia , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Cobaias , Humanos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Bainha de Mielina/patologia , Condução Nervosa/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacosRESUMO
Sepsis is commonly associated with brain dysfunction, but the underlying mechanisms remain unclear, although mitochondrial dysfunction and microvascular abnormalities have been implicated. We therefore assessed whether cerebral mitochondrial dysfunction during systemic endotoxemia in mice increased mitochondrial sensitivity to a further bioenergetic insult (hyoxemia), and whether hypothermia could improve outcome. Mice (C57bl/6) were injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg; n = 85) or saline (0.01 ml/g; n = 47). Six, 24 and 48 h later, we used confocal imaging in vivo to assess cerebral mitochondrial redox potential and cortical oxygenation in response to changes in inspired oxygen. The fraction of inspired oxygen (FiO2) at which the cortical redox potential changed was compared between groups. In a subset of animals, spontaneous hypothermia was maintained or controlled hypothermia induced during imaging. Decreasing FiO2 resulted in a more reduced cerebral redox state around veins, but preserved oxidation around arteries. This pattern appeared at a higher FiO2 in LPS-injected animals, suggesting an increased sensitivity of cortical mitochondria to hypoxemia. This increased sensitivity was accompanied by a decrease in cortical oxygenation, but was attenuated by hypothermia. These results suggest that systemic endotoxemia influences cortical oxygenation and mitochondrial function, and that therapeutic hypothermia can be protective.