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Understanding the sequence of cellular responses and their contributions to pathomorphogical changes in spinal white matter injuries is a prerequisite for developing efficient therapeutic strategies for spinal cord injury (SCI) as well as neurodegenerative and inflammatory diseases of the spinal cord such as amyotrophic lateral sclerosis and multiple sclerosis. We have developed several types of surgical procedures suitable for acute one-time and chronic recurrent in vivo multiphoton microscopy of spinal white matter [1]. Sophisticated surgical procedures were combined with transgenic mouse technology to image spinal tissue labeled with up to four fluorescent proteins (FPs) in axons, astrocytes, microglia, and blood vessels. To clearly separate the simultaneously excited FPs, spectral unmixing including iterative procedures was performed after imaging the diversely labeled spinal white matter with a custom-made 4-channel two-photon laser-scanning microscope. In our longitudinal multicellular studies of injured spinal white matter, we imaged axonal dynamics and invasion of microglia and astrocytes for a time course of over 200 days after SCI. Our methods offer ideal platforms for investigating acute and chronic cellular dynamics, cell-cell interactions, and metabolite fluctuations in health and disease as well as pharmacological manipulations in vivo.
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BACKGROUND: Rare diseases are often complex, chronic and many of them life-shortening. In Germany, healthcare for rare diseases is organized in expert centers for rare diseases. Most patients additionally have regional general practicioners and specialists for basic medical care. Thus, collaboration and information exchange between sectors is highly relevant. Our study focuses on the patient and caregiver perspective on intersectoral and interdisciplinary care between local healthcare professionals (HCPs) and centers for rare diseases in Germany. The aims were (1) to investigate patients' and caregivers' general experience of healthcare, (2) to analyse patients' and caregivers' perception of collaboration and cooperation between local healthcare professionals and expert centers for rare diseases and (3) to investigate patients' and caregivers' satisfaction with healthcare in the expert centers for rare diseases. RESULTS: In total 299 individuals of whom 176 were patients and 123 were caregivers to pediatric patients participated in a survey using a questionnaire comprising several instruments and constructs. Fifty participants were additionally interviewed using a semistructured guideline. Most patients reported to receive written information about their care, have a contact person for medical issues and experienced interdisciplinary exchange within the centers for rare diseases. Patients and caregivers in our sample were mainly satisfied with the healthcare in the centers for rare diseases. The qualitative interviews showed a rather mixed picture including experiences of uncoordinated care, low engagement and communication difficulties between professionals of different sectors. Patients reported several factors that influenced the organization and quality of healthcare e.g. engagement and health literacy in patients or engagement of HCPs. CONCLUSIONS: Our findings indicate the high relevance of transferring affected patients to specialized care as fast as possible to provide best medical treatment and increase patient satisfaction. Intersectoral collaboration should exceed written information exchange and should unburden patients of being and feeling responsible for communication between sectors and specialists. Results indicate a lack of inclusion of psychosocial aspects in routine care, which suggests opportunities for necessary improvements.
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Enfermedades Raras , Humanos , Enfermedades Raras/terapia , Alemania , Masculino , Femenino , Encuestas y Cuestionarios , Adulto , Persona de Mediana Edad , Colaboración Intersectorial , Personal de Salud/psicología , Atención a la Salud , Comunicación , Satisfacción del Paciente , Adulto Joven , Cuidadores/psicologíaRESUMEN
Hemifacial spasm (HFS) is a rare complication of idiopathic intracranial hypertension (IIH); hence, little is known about its pathophysiology and treatment options. Here, we report a 29-year-old woman with a 3-month history of IIH and HFS. Following a diagnostic and therapeutic lumbar puncture, she suffered from attacks of HFS for a few days every time she rose from a supine position. The relatively sudden drop in cerebrospinal fluid (CSF) pressure led to frequent HFS attacks, which, in contrast to the attacks in the previous months, were strictly observed only when getting up from the supine position. This observation suggests changes in CSF pressure as the likely pathophysiological mechanism responsible for HFS in IIH, rather than the absolute CSF pressure. She was then successfully treated with topiramate, a known therapeutic option in IIH, but not yet described for HFS in IIH.
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In the past, the spinal cord was considered a hard-wired network responsible for spinal reflexes and a conduit for long-range connections. This view has changed dramatically over the past few decades. It is now recognized as a plastic structure that has the potential to adapt to changing environments. While such changes occur under physiological conditions, the most dramatic alterations take place in response to pathological events. Many of the changes that occur following such pathological events are maladaptive, but some appear to help adapt to the new conditions. Although a number of studies have been devoted to elucidating the underlying mechanisms, in humans and animal models, the etiology and pathophysiology of various diseases impacting the spinal cord are still not well understood. In this review, we summarize current understanding and outstanding challenges for a number of diseases, including spinal muscular atrophy (SMA), amyotrophic laterals sclerosis (ALS), and spinal cord injury (SCI), with occasional relations to stroke. In particular, we focus on changes resulting from SCI (and stroke), and various influencing factors such as cause, site and extent of the afflicted damage.
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Esclerosis Amiotrófica Lateral , Atrofia Muscular Espinal , Traumatismos de la Médula Espinal , Accidente Cerebrovascular , Animales , Humanos , Médula Espinal , Atrofia Muscular Espinal/patología , Traumatismos de la Médula Espinal/patología , Modelos Animales de Enfermedad , Accidente Cerebrovascular/patologíaRESUMEN
A 60-year-old man suffering from recurrent attacks of yawning-fatigue-syndrome, triggered by mild exercise of his right leg since a temporary lumbar disc herniation 9 years ago, was initially treated with the oral µ-opioid-receptor agonist tilidine before each bout of exercise (see Dibaj et al. 2019 JAMA Neurology 2019;77:254). During the first few months, this treatment continuously prolonged the time without exercise-triggered yawning and fatigue. In the next few months of treatment, exercise was increased in a graded manner to alleviate the yawning-fatigue-syndrome. The number of repetitions of the physical exercises was gradually increased without taking the opioid beforehand. After several months, almost the same effort level without medication could be achieved by graded activity as before under the influence of opioid medication. Graded physical activity can thus disrupt complex pathophysiological mechanisms leading to yawning and fatigue.
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Myelin serves as an axonal insulator that facilitates rapid nerve conduction along axons. By transmission electron microscopy, a healthy myelin sheath comprises compacted membrane layers spiraling around the cross-sectioned axon. Previously we identified the assembly of septin filaments in the innermost non-compacted myelin layer as one of the latest steps of myelin maturation in the central nervous system (CNS) (Patzig et al., 2016). Here we show that loss of the cytoskeletal adaptor protein anillin (ANLN) from oligodendrocytes disrupts myelin septin assembly, thereby causing the emergence of pathological myelin outfoldings. Since myelin outfoldings are a poorly understood hallmark of myelin disease and brain aging we assessed axon/myelin-units in Anln-mutant mice by focused ion beam-scanning electron microscopy (FIB-SEM); myelin outfoldings were three-dimensionally reconstructed as large sheets of multiple compact membrane layers. We suggest that anillin-dependent assembly of septin filaments scaffolds mature myelin sheaths, facilitating rapid nerve conduction in the healthy CNS.
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Sistema Nervioso Central/metabolismo , Proteínas Contráctiles/fisiología , Vaina de Mielina/metabolismo , Septinas/metabolismo , Animales , Sistema Nervioso Central/patología , Proteínas Contráctiles/genética , Ratones , Pliegue de ProteínaRESUMEN
Multiple Sclerosis (MS) is an inflammatory demyelinating disorder in which remyelination failure contributes to persistent disability. Cholesterol is rate-limiting for myelin biogenesis in the developing CNS; however, whether cholesterol insufficiency contributes to remyelination failure in MS, is unclear. Here, we show the relationship between cholesterol, myelination and neurological parameters in mouse models of demyelination and remyelination. In the cuprizone model, acute disease reduces serum cholesterol levels that can be restored by dietary cholesterol. Concomitant with blood-brain barrier impairment, supplemented cholesterol directly supports oligodendrocyte precursor proliferation and differentiation, and restores the balance of growth factors, creating a permissive environment for repair. This leads to attenuated axon damage, enhanced remyelination and improved motor learning. Remarkably, in experimental autoimmune encephalomyelitis, cholesterol supplementation does not exacerbate disease expression. These findings emphasize the safety of dietary cholesterol in inflammatory diseases and point to a previously unrecognized role of cholesterol in promoting repair after demyelinating episodes.
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Colesterol en la Dieta/administración & dosificación , Colesterol/sangre , Esclerosis Múltiple/terapia , Proteínas de la Mielina/biosíntesis , Animales , Axones/patología , Biomarcadores/sangre , Encéfalo/citología , Encéfalo/patología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Colesterol/metabolismo , Colesterol en la Dieta/efectos adversos , Cuprizona/toxicidad , Suplementos Dietéticos , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/sangre , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/sangre , Esclerosis Múltiple/inducido químicamente , Oligodendroglía/citología , Oligodendroglía/patología , Oligodendroglía/fisiología , Cultivo Primario de Células , Células Madre/fisiologíaRESUMEN
Myelination of axons facilitates rapid impulse propagation in the nervous system. The axon/myelin-unit becomes impaired in myelin-related disorders and upon normal aging. However, the molecular cause of many pathological features, including the frequently observed myelin outfoldings, remained unknown. Using label-free quantitative proteomics, we find that the presence of myelin outfoldings correlates with a loss of cytoskeletal septins in myelin. Regulated by phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2)-levels, myelin septins (SEPT2/SEPT4/SEPT7/SEPT8) and the PI(4,5)P2-adaptor anillin form previously unrecognized filaments that extend longitudinally along myelinated axons. By confocal microscopy and immunogold-electron microscopy, these filaments are localized to the non-compacted adaxonal myelin compartment. Genetic disruption of these filaments in Sept8-mutant mice causes myelin outfoldings as a very specific neuropathology. Septin filaments thus serve an important function in scaffolding the axon/myelin-unit, evidently a late stage of myelin maturation. We propose that pathological or aging-associated diminishment of the septin/anillin-scaffold causes myelin outfoldings that impair the normal nerve conduction velocity.
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Sistema Nervioso Central/fisiología , Proteínas Contráctiles/metabolismo , Vaina de Mielina/metabolismo , Conducción Nerviosa , Septinas/metabolismo , Animales , Sistema Nervioso Central/química , Citoesqueleto/metabolismo , Técnicas de Inactivación de Genes , Marcación de Gen , Ratones , Microscopía Confocal , Microscopía Inmunoelectrónica , Fibras Nerviosas Mielínicas/química , Fibras Nerviosas Mielínicas/fisiología , Multimerización de Proteína , Proteoma/análisis , Proteómica , Septinas/genéticaRESUMEN
Oligodendrocytes make myelin and support axons metabolically with lactate. However, it is unknown how glucose utilization and glycolysis are adapted to the different axonal energy demands. Spiking axons release glutamate and oligodendrocytes express NMDA receptors of unknown function. Here we show that the stimulation of oligodendroglial NMDA receptors mobilizes glucose transporter GLUT1, leading to its incorporation into the myelin compartment in vivo. When myelinated optic nerves from conditional NMDA receptor mutants are challenged with transient oxygen-glucose deprivation, they show a reduced functional recovery when returned to oxygen-glucose but are indistinguishable from wild-type when provided with oxygen-lactate. Moreover, the functional integrity of isolated optic nerves, which are electrically silent, is extended by preincubation with NMDA, mimicking axonal activity, and shortened by NMDA receptor blockers. This reveals a novel aspect of neuronal energy metabolism in which activity-dependent glutamate release enhances oligodendroglial glucose uptake and glycolytic support of fast spiking axons.
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Axones/metabolismo , Metabolismo Energético/fisiología , Glucosa/metabolismo , Oligodendroglía/metabolismo , Nervio Óptico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Transportador de Glucosa de Tipo 1/metabolismo , Ratones Transgénicos , Vaina de Mielina/metabolismo , Oxígeno/metabolismoRESUMEN
OBJECTIVES: In the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS), a selective degeneration of fast-fatigable motor units and consequently an early decline of contractile force in individual fast-twitch muscles have been observed in the preclinical stage. However, most human muscles include fast and slow motor units. Gastrocnemius-soleus group (GS) contains such a mixture of units. METHODS: We have investigated changes in the mechanical properties of GS at different SOD1G93A stages in mice. For this purpose, the tibial nerve was repetitively stimulated with rectangular pulses and the force of GS twitches was recorded using a strain gauge fixed to the Achilles tendon. RESULTS: Isometric and tetanic force were attenuated but not before the first clinical signs developed. However, already at preclinical stages, single twitches showed a slower decay compared to control. Consequently, fusion of GS twitches occurred at lower stimulus rates. Furthermore, already preclinically, the temporal course of successive twitch amplitudes changed during repetitive stimulation at increasing rates. The peak amplitudes as well as the potentiation following decay (fatigue) were lower in preclinical mice than in control. DISCUSSION: The time-lapse analysis of the contractile pattern as well as of the twitch configuration of the mixed muscle GS have revealed distinctive differences between wild-type controls and preclinical SOD1G93A mice. It would be of interest to know whether these preclinical changes are also detectable in ALS patients.
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Esclerosis Amiotrófica Lateral/fisiopatología , Contracción Muscular/fisiología , Músculo Esquelético/fisiopatología , Tendón Calcáneo/fisiopatología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Estimulación Eléctrica , Humanos , Ratones Transgénicos , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Nervio Tibial/fisiopatologíaRESUMEN
Neuregulin-1 (NRG1) gene variants are associated with increased genetic risk for schizophrenia. It is unclear whether risk haplotypes cause elevated or decreased expression of NRG1 in the brains of schizophrenia patients, given that both findings have been reported from autopsy studies. To study NRG1 functions in vivo, we generated mouse mutants with reduced and elevated NRG1 levels and analyzed the impact on cortical functions. Loss of NRG1 from cortical projection neurons resulted in increased inhibitory neurotransmission, reduced synaptic plasticity, and hypoactivity. Neuronal overexpression of cysteine-rich domain (CRD)-NRG1, the major brain isoform, caused unbalanced excitatory-inhibitory neurotransmission, reduced synaptic plasticity, abnormal spine growth, altered steady-state levels of synaptic plasticity-related proteins, and impaired sensorimotor gating. We conclude that an "optimal" level of NRG1 signaling balances excitatory and inhibitory neurotransmission in the cortex. Our data provide a potential pathomechanism for impaired synaptic plasticity and suggest that human NRG1 risk haplotypes exert a gain-of-function effect.
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Neurregulina-1/metabolismo , Plasticidad Neuronal , Células Piramidales/fisiología , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/fisiología , Movimiento Celular , Condicionamiento Psicológico , Espinas Dendríticas/fisiología , Miedo , Femenino , Expresión Génica , Interneuronas/fisiología , Masculino , Ratones Transgénicos , Red Nerviosa , Neurregulina-1/genética , Transmisión SinápticaRESUMEN
Investigation and interpretation of defective motor circuitries in transgenic mice required further basic results from wild-type mice. Therefore, we investigated the lumbar motor reflex pattern in anaesthetised mice using intracellular motoneuronal recording and monosynaptic reflex testing. Thresholds and latencies in mice were similar to those in cats: thresholds for monosynaptic (group I) EPSPs were slightly above 1T (T=threshold for the lowest threshold fibres), around 1.5T for group II EPSPs and above 10T for group III EPSPs; group I EPSPs were maximal with a stimulus strength around 2T, group II EPSPs were maximal with 5-8T; latencies to the group I incoming volley were below 1ms for monosynaptic group I EPSPs, around 3ms for polysynaptic group II EPSPs and above 4ms for polysynaptic group III EPSPs. In contrast to reflex actions in the cat, monosynaptic gastrocnemius-soleus reflexes were facilitated by conditioning stimulation of the peroneal, sural and tibial nerves, i.e. by a variety of different, probably flexor reflex afferents. This facilitation persisted after high lumbar spinalisation indicating an independency to supraspinal influences. Nociceptive muscle afferents facilitated the peroneal monosynaptic reflex while nociceptive cutaneous afferents from the foot sole inhibited the ipsilateral but facilitated the contralateral peroneal reflex.
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Vías Aferentes/fisiología , Neuronas Motoras/fisiología , Neuronas Aferentes/fisiología , Reflejo/fisiología , Médula Espinal/fisiología , Animales , Gatos , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/fisiología , Pie/inervación , Región Lumbosacra , RatonesRESUMEN
Mutations in SOD1 cause hereditary variants of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous, with toxicity deriving also from glia. In particular, microglia contribute to disease progression. Methylene blue (MB) inhibits the effect of nitric oxide, which mediates microglial responses to injury. In vivo 2P-LSM imaging was performed in ALS-linked transgenic SOD1(G93A) mice to investigate the effect of MB on microglia-mediated inflammation in the spinal cord. Local superfusion of the lateral spinal cord with MB inhibited the microglial reaction directed at a laser-induced axon transection in control and SOD1(G93A) mice. In vitro, MB at high concentrations inhibited cytokine and chemokine release from microglia of control and advanced clinical SOD1(G93A) mice. Systemic MB-treatment of SOD1(G93A) mice at early preclinical stages significantly delayed disease onset and motor dysfunction. However, an increase of MB dose had no additional effect on disease progression; this was unexpected in view of the local anti-inflammatory effects. Furthermore, in vivo imaging of systemically MB-treated mice also showed no alterations of microglia activity in response to local lesions. Thus although systemic MB treatment had no effect on microgliosis, instead, its use revealed an important influence on motor neuron survival as indicated by an increased number of lumbar anterior horn neurons present at the time of disease onset. Thus, potentially beneficial effects of locally applied MB on inflammatory events contributing to disease progression could not be reproduced in SOD1(G93A) mice via systemic administration, whereas systemic MB application delayed disease onset via neuroprotection.
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Esclerosis Amiotrófica Lateral/patología , Azul de Metileno/farmacología , Microglía/efectos de los fármacos , Microglía/patología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Animales Modificados Genéticamente , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Microglía/metabolismo , Actividad Motora/efectos de los fármacos , Superóxido Dismutasa-1 , Factores de TiempoRESUMEN
Duplication of PLP1 (proteolipid protein gene 1) and the subsequent overexpression of the myelin protein PLP (also known as DM20) in oligodendrocytes is the most frequent cause of Pelizaeus-Merzbacher disease (PMD), a fatal leukodystrophy without therapeutic options. PLP binds cholesterol and is contained within membrane lipid raft microdomains. Cholesterol availability is the rate-limiting factor of central nervous system myelin synthesis. Transgenic mice with extra copies of the Plp1 gene are accurate models of PMD. Dysmyelination followed by demyelination, secondary inflammation and axon damage contribute to the severe motor impairment in these mice. The finding that in Plp1-transgenic oligodendrocytes, PLP and cholesterol accumulate in late endosomes and lysosomes (endo/lysosomes), prompted us to further investigate the role of cholesterol in PMD. Here we show that cholesterol itself promotes normal PLP trafficking and that dietary cholesterol influences PMD pathology. In a preclinical trial, PMD mice were fed a cholesterol-enriched diet. This restored oligodendrocyte numbers and ameliorated intracellular PLP accumulation. Moreover, myelin content increased, inflammation and gliosis were reduced and motor defects improved. Even after onset of clinical symptoms, cholesterol treatment prevented disease progression. Dietary cholesterol did not reduce Plp1 overexpression but facilitated incorporation of PLP into myelin membranes. These findings may have implications for therapeutic interventions in patients with PMD.
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Colesterol en la Dieta/administración & dosificación , Colesterol en la Dieta/uso terapéutico , Conducta Alimentaria , Enfermedad de Pelizaeus-Merzbacher/dietoterapia , Animales , Colesterol en la Dieta/farmacología , Regulación de la Expresión Génica , Ratones , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/patología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Oligodendroglía/patología , Nervio Óptico/efectos de los fármacos , Nervio Óptico/patología , Nervio Óptico/ultraestructura , Enfermedad de Pelizaeus-Merzbacher/genética , Enfermedad de Pelizaeus-Merzbacher/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Factores de TiempoRESUMEN
We demonstrated superresolution optical microscopy in a living higher animal. Stimulated emission depletion (STED) fluorescence nanoscopy reveals neurons in the cerebral cortex of a mouse with <70-nanometer resolution. Dendritic spines and their subtle changes can be observed at their relevant scales over extended periods of time.
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Espinas Dendríticas/fisiología , Espinas Dendríticas/ultraestructura , Microscopía Fluorescente/métodos , Corteza Somatosensorial/ultraestructura , Animales , Fluorescencia , Proteínas Luminiscentes , Ratones , Microscopía Fluorescente/instrumentación , NanotecnologíaRESUMEN
To study the function of individual neurons that are embedded in a complex neural network is difficult in mice. Conditional mutagenesis permits the spatiotemporal control of gene expression including the ablation of cells by toxins. To direct expression of a tamoxifen-inducible variant of Cre recombinase (CreERT2) selectively to cortical neurons, we replaced the coding region of the murine Nex1 gene by CreERT2 cDNA via homologous recombination in embryonic stem cells. When injected with tamoxifen, adult NEX-CreERT2 mice induced reporter gene expression exclusively in projection neurons of the neocortex and hippocampus. By titrating the tamoxifen dosage, we achieved recombination in single cells, which allowed multiphoton imaging of neocortical neurons in live mice. When hippocampal projection neurons were genetically ablated by induced expression of diphteria toxin, within 20 days the inflammatory response included the infiltration of CD3+ T cells. This marks a striking difference from similar studies, in which dying oligodendrocytes failed to recruit cells of the adaptive immune system.