RESUMO
BACKGROUND: Rapidly determining the causes of a depressed level of consciousness (DLOC) including coma is a common clinical challenge. Quantitative analysis of the electroencephalogram (EEG) has the potential to improve DLOC assessment by providing readily deployable, temporally detailed characterization of brain activity in such patients. While used commonly for seizure detection, EEG-based assessment of DLOC etiology is less well-established. As a first step towards etiological diagnosis, we sought to distinguish focal and diffuse causes of DLOC through assessment of temporal dynamics within EEG signals. METHODS: We retrospectively analyzed EEG recordings from 40 patients with DLOC with consensus focal or diffuse culprit pathology. For each recording, we performed a suite of time-series analyses, then used a statistical framework to identify which analyses (features) could be used to distinguish between focal and diffuse cases. RESULTS: Using cross-validation approaches, we identified several spectral and non-spectral EEG features that were significantly different between DLOC patients with focal vs. diffuse etiologies, enabling EEG-based classification with an accuracy of 76%. CONCLUSIONS: Our findings suggest that DLOC due to focal vs. diffuse injuries differ along several electrophysiological parameters. These results may form the basis of future classification strategies for DLOC and coma that are more etiologically-specific and therefore therapeutically-relevant.
Assuntos
Coma/etiologia , Transtornos da Consciência/etiologia , Eletroencefalografia/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
The great majority of acute brain injury results from trauma or from disorders of the cerebrovasculature, i.e. ischaemic stroke or haemorrhage. These injuries are characterized by an initial insult that triggers a cascade of injurious cellular processes. The nature of these processes in spontaneous intracranial haemorrhage is poorly understood. Subarachnoid haemorrhage, a particularly deadly form of intracranial haemorrhage, shares key pathophysiological features with traumatic brain injury including exposure to a sudden pressure pulse. Here we provide evidence that axonal injury, a signature characteristic of traumatic brain injury, is also a prominent feature of experimental subarachnoid haemorrhage. Using histological markers of membrane disruption and cytoskeletal injury validated in analyses of traumatic brain injury, we show that axonal injury also occurs following subarachnoid haemorrhage in an animal model. Consistent with the higher prevalence of global as opposed to focal deficits after subarachnoid haemorrhage and traumatic brain injury in humans, axonal injury in this model is observed in a multifocal pattern not limited to the immediate vicinity of the ruptured artery. Ultrastructural analysis further reveals characteristic axonal membrane and cytoskeletal changes similar to those associated with traumatic axonal injury. Diffusion tensor imaging, a translational imaging technique previously validated in traumatic axonal injury, from these same specimens demonstrates decrements in anisotropy that correlate with histological axonal injury and functional outcomes. These radiological indicators identify a fibre orientation-dependent gradient of axonal injury consistent with a barotraumatic mechanism. Although traumatic and haemorrhagic acute brain injury are generally considered separately, these data suggest that a signature pathology of traumatic brain injury-axonal injury-is also a functionally significant feature of subarachnoid haemorrhage, raising the prospect of common diagnostic, prognostic, and therapeutic approaches to these conditions.
Assuntos
Axônios/patologia , Lesões Encefálicas/diagnóstico , Lesões Encefálicas/etiologia , Hemorragia Subaracnóidea/complicações , Peptídeos beta-Amiloides/metabolismo , Animais , Axônios/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/patologia , Imagem de Tensor de Difusão , Modelos Animais de Doenças , Imageamento por Ressonância Magnética , Masculino , Transtornos Mentais/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas de Neurofilamentos/metabolismo , Estatística como Assunto , Hemorragia Subaracnóidea/patologia , Fatores de Tempo , UltrassonografiaRESUMO
Background: Inflammation contributes to morbidity following subarachnoid hemorrhage (SAH). Transauricular vagus nerve stimulation (taVNS) offers a noninvasive approach to target the inflammatory response following SAH. Methods: In this prospective, triple-blinded, randomized, controlled trial, twenty-seven patients were randomized to taVNS or sham stimulation. Blood and cerebrospinal fluid (CSF) were collected to quantify inflammatory markers. Cerebral vasospasm severity and functional outcomes (modified Rankin Scale, mRS) were analyzed. Results: No adverse events occurred. Radiographic vasospasm was significantly reduced (p = 0.018), with serial vessel caliber measurements demonstrating a more rapid return to normal than sham (p < 0.001). In the taVNS group, TNF-α was significantly reduced in both plasma (days 7 and 10) and CSF (day 13); IL-6 was also significantly reduced in plasma (day 4) and CSF (day 13) (p < 0.05). Patients receiving taVNS had higher rates of favorable outcomes at discharge (38.4% vs 21.4%) and first follow-up (76.9% vs 57.1%), with significant improvement from admission to first follow-up (p = 0.014), unlike the sham group (p = 0.18). The taVNS group had a significantly lower rate of discharge to skilled nursing facility or hospice (p = 0.04). Conclusion: taVNS is a non-invasive method of neuro- and systemic immunomodulation. This trial supports that taVNS following SAH can mitigate the inflammatory response, reduce radiographic vasospasm, and potentially improve functional and neurological outcomes. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT04557618.
RESUMO
Background: Inflammation has been implicated in driving the morbidity associated with subarachnoid hemorrhage (SAH). Despite understanding the important role of inflammation in morbidity following SAH, there is no current effective way to modulate this deleterious response. There is a critical need for a novel approach to immunomodulation that can be safely, rapidly, and effectively deployed in SAH patients. Vagus nerve stimulation (VNS) provides a non-pharmacologic approach to immunomodulation, with prior studies demonstrating VNS can reduce systemic inflammatory markers, and VNS has had early success treating inflammatory conditions such as arthritis, sepsis, and inflammatory bowel diseases. The aim of the Non-invasive Auricular Vagus nerve stimulation for Subarachnoid Hemorrhage (NAVSaH) trial is to translate the use of non-invasive transcutaneous auricular VNS (taVNS) to spontaneous SAH, with our central hypothesis being that implementing taVNS in the acute period following spontaneous SAH attenuates the expected inflammatory response to hemorrhage and curtails morbidity associated with inflammatory-mediated clinical endpoints. Materials and methods: The overall objectives for the NAHSaH trial are to 1) Define the impact that taVNS has on SAH-induced inflammatory markers in the plasma and cerebrospinal fluid (CSF), 2) Determine whether taVNS following SAH reduces radiographic vasospasm, and 3) Determine whether taVNS following SAH reduces chronic hydrocephalus. Following presentation to a single enrollment site, enrolled SAH patients are randomly assigned twice daily treatment with either taVNS or sham stimulation for the duration of their intensive care unit stay. Blood and CSF are drawn before initiation of treatment sessions, and then every three days during a patient's hospital stay. Primary endpoints include change in the inflammatory cytokine TNF-α in plasma and cerebrospinal fluid between day 1 and day 13, rate of radiographic vasospasm, and rate of requirement for long-term CSF diversion via a ventricular shunt. Secondary outcomes include exploratory analyses of a panel of additional cytokines, number and type of hospitalized acquired infections, duration of external ventricular drain in days, interventions required for vasospasm, continuous physiology data before, during, and after treatment sessions, hospital length of stay, intensive care unit length of stay, and modified Rankin Scale score (mRS) at admission, discharge, and each at follow-up appointment for up to two years following SAH. Discussion: Inflammation plays a central role in morbidity following SAH. This NAVSaH trial is innovative because it diverges from the pharmacologic status quo by harnessing a novel non-invasive neuromodulatory approach and its known anti-inflammatory effects to alter the pathophysiology of SAH. The investigation of a new, effective, and rapidly deployable intervention in SAH offers a new route to improve outcomes following SAH. Trial registration: Clinical Trials Registered, NCT04557618. Registered on September 21, 2020, and the first patient was enrolled on January 4, 2021.
RESUMO
Introduction: Subarachnoid hemorrhage (SAH) is characterized by intense central inflammation, leading to substantial post-hemorrhagic complications such as vasospasm and delayed cerebral ischemia. Given the anti-inflammatory effect of transcutaneous auricular vagus nerve stimulation (taVNS) and its ability to promote brain plasticity, taVNS has emerged as a promising therapeutic option for SAH patients. 3,10,13 However, the effects of taVNS on cardiovascular dynamics in critically ill patients like those with SAH have not yet been investigated. Given the association between cardiac complications and elevated risk of poor clinical outcomes after SAH, it is essential to characterize the cardiovascular effects of taVNS to ensure this approach is safe in this fragile population 5 . Therefore, we assessed the impact of both acute taVNS and repetitive taVNS on cardiovascular function in this study. Methods: In this randomized clinical trial, 24 SAH patients were assigned to either a taVNS treatment or a Sham treatment group. During their stay in the intensive care unit, we monitored patient electrocardiogram (ECG) readings and vital signs. We compared long-term changes in heart rate, heart rate variability, QT interval, and blood pressure between the two groups. Additionally, we assessed the effects of acute taVNS by comparing cardiovascular metrics before, during, and after the intervention. We also explored rapidly responsive cardiovascular biomarkers in patients exhibiting clinical improvement. Results: We found that repetitive taVNS did not significantly alter heart rate, corrected QT interval, blood pressure, or intracranial pressure. However, taVNS increased overall heart rate variability and parasympathetic activity from 5-10 days after initial treatment, as compared to the sham treatment. Acutely, taVNS increased heart rate, blood pressure, and peripheral perfusion index without affecting the corrected QT interval, intracranial pressure, or heart rate variability. The acute post-treatment elevation in heart rate was more pronounced in patients who experienced a decrease of more than 1 point in their Modified Rankin Score at the time of discharge. Conclusions: Our study found that taVNS treatment did not induce adverse cardiovascular effects, such as bradycardia or QT prolongation, supporting its development as a safe immunomodulatory treatment approach for SAH patients. The observed acute increase in heart rate after taVNS treatment may serve as a biomarker for SAH patients who could derive greater benefit from this treatment. Trial registration: NCT04557618.
RESUMO
Introduction: Traumatic optic neuropathy (TON) is the optic nerve injury secondary to brain trauma leading to visual impairment and vision loss. Current clinical visual function assessments often fail to detect TON due to slow disease progression and clinically silent lesions resulting in potentially delayed or missed treatment in patients with traumatic brain injury (TBI). Methods: Diffusion basis spectrum imaging (DBSI) is a novel imaging modality that can potentially fill this diagnostic gap. Twenty-two, 16-week-old, male mice were equally divided into a sham or TBI (induced by moderate Closed-Head Impact Model of Engineered Rotational Acceleration device) group. Briefly, mice were anesthetized with isoflurane (5% for 2.5 min followed by 2.5% maintenance during injury induction), had a helmet placed over the head, and were placed in a holder prior to a 2.1-joule impact. Serial visual acuity (VA) assessments, using the Virtual Optometry System, and DBSI scans were performed in both groups of mice. Immunohistochemistry (IHC) and histological analysis of optic nerves was also performed after in vivo MRI. Results: VA of the TBI mice showed unilateral or bilateral impairment. DBSI of the optic nerves exhibited bilateral involvement. IHC results of the optic nerves revealed axonal loss, myelin injury, axonal injury, and increased cellularity in the optic nerves of the TBI mice. Increased DBSI axon volume, decreased DBSI λ||, and elevated DBSI restricted fraction correlated with decreased SMI-312, decreased SMI-31, and increased DAPI density, respectively, suggesting that DBSI can detect coexisting pathologies in the optic nerves of TBI mice. Conclusion: DBSI provides an imaging modality capable of detecting subclinical changes of indirect TON in TBI mice.
RESUMO
Immunofluorescence labeling and microscopy offer a highly specific means to visualize proteins or other molecular species in a sample by labeling target antigens with fluorescent probes. These fluorescent probes can then be visualized using a fluorescence microscope, allowing their relative spatial relationships to be determined. Due to spectral overlap of common fluorophores, however, it can be challenging to analyze more than three antigens in a single sample with standard imaging approaches. This article describes multiplexed labeling and imaging of four target antigens through the use of a long-Stokes-shift fluorophore-a fluorophore with an unusually large gap between its excitation and emission maxima-in tandem with three conventional fluorophores. This combination allows for multiplexed imaging of four antigens in a single sample with excellent spectral discrimination suitable for sensitive analyses using standard imaging hardware. Particular advantages of this approach are its flexibility in terms of target antigens and the lack of any specialized procedures, reagents, or equipment beyond the commercially available labeling reagent coupled to the long-Stokes-shift fluorophore. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Four-probe immunofluorescence labeling Basic Protocol 2: Four-probe immunofluorescence imaging.
Assuntos
Corantes Fluorescentes , Proteínas , Ionóforos , Microscopia de FluorescênciaRESUMO
Alzheimer's disease (AD) is tightly correlated with synapse loss in vulnerable brain regions. It is assumed that specific molecular entities such as Aß and tau cause synapse loss in AD, yet unbiased screens for synaptotoxic activities have not been performed. Here, we performed size exclusion chromatography on soluble human brain homogenates from AD cases, high pathology non-demented controls, and low pathology age-matched controls using our novel high content primary cultured neuron-based screening assay. Both presynaptic and postsynaptic toxicities were elevated in homogenates from AD cases and high pathology non-demented controls to a similar extent, with more modest synaptotoxic activities in homogenates from low pathology normal controls. Surprisingly, synaptotoxic activities were found in size fractions peaking between the 17-44 kDa size standards that did not match well with Aß and tau immunoreactive species in these homogenates. The fractions containing previously identified high molecular weight soluble amyloid beta aggregates/"oligomers" were non-toxic in this assay. Furthermore, immunodepletion of Aß and tau did not reduce synaptotoxic activity. This result contrasts with previous findings involving the same methods applied to 3xTg-AD mouse brain extracts. The nature of the synaptotoxic species has not been identified. Overall, our data indicates one or more potential Aß and tau independent synaptotoxic activities in human AD brain homogenates. This result aligns well with the key role of synaptic loss in the early cognitive decline and may provide new insight into AD pathophysiology.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Neurônios/patologia , Sinapses/patologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Estudos de Casos e Controles , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Camundongos , Neurônios/metabolismo , Cultura Primária de Células , Sinapses/metabolismo , Proteínas tau/metabolismoRESUMO
Synapses are crucial to brain function and frequent disease targets, but current analysis methods cannot report on individual synaptic components in situ or present barriers to widespread adoption. SEQUIN was developed to address this challenge. SEQUIN utilizes a widely available super-resolution platform in tandem with image processing and analysis to quantify synaptic loci over large regions of brain and characterize their molecular and nanostructural properties at the individual and population level. This protocol describes quantification of synaptic loci using SEQUIN. For additional details on the use and execution of this protocol, please refer to Sauerbeck et al. (2020).
Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Processamento de Imagem Assistida por Computador , Sinapses/metabolismo , Animais , CamundongosRESUMO
BACKGROUND: CNIH3 is an AMPA receptor (AMPAR) auxiliary protein prominently expressed in the dorsal hippocampus (dHPC), a region that plays a critical role in spatial memory and synaptic plasticity. However, the effects of CNIH3 on AMPAR-dependent synaptic function and behavior have not been investigated. METHODS: We assessed a gain-of-function model of Cnih3 overexpression in the dHPC and generated and characterized a line of Cnih3-/- C57BL/6 mice. We assessed spatial memory through behavioral assays, protein levels of AMPAR subunits and synaptic proteins by immunoblotting, and long-term potentiation in electrophysiological recordings. We also utilized a super-resolution imaging workflow, SEQUIN (Synaptic Evaluation and Quantification by Imaging of Nanostructure), for analysis of nanoscale synaptic connectivity in the dHPC. RESULTS: Overexpression of Cnih3 in the dHPC improved short-term spatial memory in female mice but not in male mice. Cnih3-/- female mice exhibited weakened short-term spatial memory, reduced dHPC synapse density, enhanced expression of calcium-impermeable AMPAR (GluA2-containing) subunits in synaptosomes, and attenuated long-term potentiation maintenance compared with Cnih3+/+ control mice; Cnih3-/- males were unaffected. Further investigation revealed that deficiencies in spatial memory and changes in AMPAR composition and synaptic plasticity were most pronounced during the metestrus phase of the estrous cycle in female Cnih3-/- mice. CONCLUSIONS: This study identified a novel effect of sex and estrous on CNIH3's role in spatial memory and synaptic plasticity. Manipulation of CNIH3 unmasked sexually dimorphic effects on spatial memory, synaptic function, AMPAR composition, and hippocampal plasticity. These findings reinforce the importance of considering sex as a biological variable in studies of memory and hippocampal synaptic function.
Assuntos
Caracteres Sexuais , Memória Espacial , Animais , Feminino , Hipocampo/metabolismo , Potenciação de Longa Duração , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Transmissão SinápticaRESUMO
The pathogenic mechanism by which dominant mutations in VCP cause multisystem proteinopathy (MSP), a rare neurodegenerative disease that presents as fronto-temporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), remains unclear. To explore this, we inactivate VCP in murine postnatal forebrain neurons (VCP conditional knockout [cKO]). VCP cKO mice have cortical brain atrophy, neuronal loss, autophago-lysosomal dysfunction, and TDP-43 inclusions resembling FTLD-TDP pathology. Conditional expression of a single disease-associated mutation, VCP-R155C, in a VCP null background similarly recapitulates features of VCP inactivation and FTLD-TDP, suggesting that this MSP mutation is hypomorphic. Comparison of transcriptomic and proteomic datasets from genetically defined patients with FTLD-TDP reveal that progranulin deficiency and VCP insufficiency result in similar profiles. These data identify a loss of VCP-dependent functions as a mediator of FTLD-TDP and reveal an unexpected biochemical similarity with progranulin deficiency.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Degeneração Lobar Frontotemporal/patologia , Neurônios/metabolismo , Proteína com Valosina/metabolismo , Idoso , Alelos , Animais , Atrofia , Autofagossomos/metabolismo , Comportamento Animal , Encéfalo/patologia , Degeneração Lobar Frontotemporal/genética , Gliose/patologia , Humanos , Lisossomos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Degeneração Neural/patologia , Neurônios/patologia , Proteômica , Transcriptoma/genéticaRESUMO
In both neurons and muscle fibers, specific mRNAs are concentrated beneath and locally translated at synaptic sites. At the skeletal neuromuscular junction, all synaptic RNAs identified to date encode synaptic components. Using microarrays, we compared RNAs in synapse-rich and -free regions of muscles, thereby identifying transcripts that are enriched near synapses and that encode soluble membrane and nuclear proteins. One gene product, LL5beta, binds to both phosphoinositides and a cytoskeletal protein, filamin, one form of which is concentrated at synaptic sites. LL5beta is itself associated with the cytoplasmic face of the postsynaptic membrane; its highest levels border regions of highest acetylcholine receptor (AChR) density, which suggests a role in "corraling" AChRs. Consistent with this idea, perturbing LL5beta expression in myotubes inhibits AChR aggregation. Thus, a strategy designed to identify novel synaptic components led to identification of a protein required for assembly of the postsynaptic apparatus.
Assuntos
Interleucina-5/biossíntese , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Junção Neuromuscular/fisiologia , Animais , Diferenciação Celular/fisiologia , Proteínas Contráteis/metabolismo , Filaminas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Camundongos , Proteínas dos Microfilamentos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositóis/metabolismo , Ligação Proteica , RNA Mensageiro/biossíntese , Receptores Colinérgicos/metabolismoRESUMO
Accurate quantification of synaptic changes is essential for understanding the molecular mechanisms of synaptogenesis, synaptic plasticity, and synaptic toxicity. Here we demonstrate a robust high-content imaging method for the assessment of synaptic changes and apply the method to brain homogenates from an Alzheimer's disease mouse model. Our method uses serial imaging of endogenous fluorescent labeled presynaptic VAMP2 and postsynaptic PSD95 in long-term cultured live primary neurons in 96 well microplates, and uses automatic image analysis to quantify the number of colocalized mature synaptic puncta for the assessment of synaptic changes in live neurons. As a control, we demonstrated that our synaptic puncta assay is at least 10-fold more sensitive to the toxic effects of glutamate than the MTT assay. Using our assay, we have compared synaptotoxic activities in size-exclusion chromatography fractioned protein samples from 3xTg-AD mouse model brain homogenates. Multiple synaptotoxic activities were found in high and low molecular weight fractions. Amyloid-beta immunodepletion alleviated some but not all of the synaptotoxic activities. Although the biochemical entities responsible for the synaptotoxic activities have yet to be determined, these proof-of-concept results demonstrate that this novel assay may have many potential mechanistic and therapeutic applications.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Neurônios/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Modelos Animais de Doenças , Proteína 4 Homóloga a Disks-Large/genética , Camundongos , Camundongos Knockout , Neurônios/patologia , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
Alzheimer's disease (AD) is characterized by plaques containing amyloid-ß (Aß) and neurofibrillary tangles composed of aggregated, hyperphosphorylated tau. Beyond tau and Aß, evidence suggests that microglia play an important role in AD pathogenesis. Rare variants in the microglia-expressed triggering receptor expressed on myeloid cells 2 (TREM2) gene increase AD risk 2- to 4-fold. It is likely that these TREM2 variants increase AD risk by decreasing the response of microglia to Aß and its local toxicity. However, neocortical Aß pathology occurs many years before neocortical tau pathology in AD. Thus, it will be important to understand the role of TREM2 in the context of tauopathy. We investigated the impact of the AD-associated TREM2 variant (R47H) on tau-mediated neuropathology in the PS19 mouse model of tauopathy. We assessed PS19 mice expressing human TREM2CV (common variant) or human TREM2R47H. PS19-TREM2R47H mice had significantly attenuated brain atrophy and synapse loss versus PS19-TREM2CV mice. Gene expression analyses and CD68 immunostaining revealed attenuated microglial reactivity in PS19-TREM2R47H versus PS19-TREM2CV mice. There was also a decrease in phagocytosis of postsynaptic elements by microglia expressing TREM2R47H in the PS19 mice and in human AD brains. These findings suggest that impaired TREM2 signaling reduces microglia-mediated neurodegeneration in the setting of tauopathy.
Assuntos
Doença de Alzheimer , Gliose , Glicoproteínas de Membrana , Mutação de Sentido Incorreto , Receptores Imunológicos , Proteínas tau , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Substituição de Aminoácidos , Animais , Gliose/genética , Gliose/metabolismo , Gliose/patologia , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microglia/metabolismo , Microglia/patologia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
The brain's complex microconnectivity underlies its computational abilities and vulnerability to injury and disease. It has been challenging to illuminate the features of this synaptic network due to the small size and dense packing of its elements. Here, we describe a rapid, accessible super-resolution imaging and analysis workflow-SEQUIN-that quantifies central synapses in human tissue and animal models, characterizes their nanostructural and molecular features, and enables volumetric imaging of mesoscale synaptic networks without the production of large histological arrays. Using SEQUIN, we identify cortical synapse loss resulting from diffuse traumatic brain injury, a highly prevalent connectional disorder. Similar synapse loss is observed in three murine models of Alzheimer-related neurodegeneration, where SEQUIN mesoscale mapping identifies regional synaptic vulnerability. These results establish an easily implemented and robust nano-to-mesoscale synapse quantification and characterization method. They furthermore identify a shared mechanism-synaptopathy-between Alzheimer neurodegeneration and its best-established epigenetic risk factor, brain trauma.
Assuntos
Lesões Encefálicas Traumáticas/patologia , Sistema Nervoso Central/diagnóstico por imagem , Nanoestruturas/ultraestrutura , Vias Neurais/diagnóstico por imagem , Vias Neurais/ultraestrutura , Neuroimagem/métodos , Sinapses/ultraestrutura , Animais , Mapeamento Encefálico , Sistema Nervoso Central/ultraestrutura , Córtex Cerebral/patologia , Humanos , Mamíferos , CamundongosRESUMO
As the mammalian neuromuscular junction matures, its acetylcholine receptor (AChR)-rich postsynaptic apparatus is transformed from an oval plaque into a pretzel-shaped array of branches that precisely mirrors the branching pattern of the motor nerve terminal. Although the nerve has been believed to direct postsynaptic maturation, we report here that myotubes cultured aneurally on matrix-coated substrates form elaborately branched AChR-rich domains remarkably similar to those seen in vivo. These domains share several characteristics with the mature postsynaptic apparatus, including colocalization of multiple postsynaptic markers, clustering of subjacent myonuclei, and dependence on the muscle-specific kinase and rapsyn for their formation. Time-lapse imaging showed that branched structures arise from plaques by formation and fusion of AChR-poor perforations through a series of steps mirroring that seen in vivo. Multiple fluorophore imaging showed that growth occurs by circumferential, asymmetric addition of AChRs. Analysis in vivo revealed similar patterns of AChR addition during normal development. These results reveal the sequence of steps by which a topologically complex domain forms on a cell and suggest an unexpected nerve-independent role for the postsynaptic cell in generating this topological complexity.
Assuntos
Junção Neuromuscular/fisiologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Sinapses/fisiologia , Animais , Linhagem Celular , Camundongos , Camundongos Transgênicos , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura , Junção Neuromuscular/ultraestrutura , Receptores Colinérgicos/fisiologia , Sinapses/ultraestruturaRESUMO
Studies of the vertebrate skeletal neuromuscular junction led to an influential model of how neurotransmitter receptors accumulate in the postsynaptic membrane. In this model, motor axons organize postsynaptic development by secreting neuregulin to induce acetylcholine receptor gene transcription in specialized subsynaptic nuclei, agrin to cluster diffuse receptors in the postsynaptic membrane, and acetylcholine to evoke electrical activity that promotes synaptic maturation. However, new studies in this area have first, demonstrated that axons sometimes innervate pre-existing receptor clusters; second, recast the roles of agrin and neuregulin; third, revealed early effects of neurotransmission; fourth, questioned the role of subsynaptic myonuclei; fifth, shown that elaborately-branched postsynaptic structures can form aneurally; and sixth, raised the possibility that neurotransmitter affects receptor type as well as distribution. These recent studies challenge the widely-held paradigms, although not the results that led to them, and suggest a new model for neuromuscular synaptogenesis.
Assuntos
Junção Neuromuscular/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Modelos Neurológicos , Receptores Colinérgicos/fisiologia , Sinapses/fisiologiaRESUMO
OBJECTIVE: We devise a data-driven framework to assess the level of consciousness in etiologically heterogeneous comatose patients using intrinsic dynamical changes of resting-state Electroencephalogram (EEG) signals. METHODS: EEG signals were collected from 54 comatose patients (GCSâ¯â©½â¯8) and 20 control patients (GCSâ¯>â¯8). We analyzed the EEG signals using a new technique, termed Intrinsic Network Reactivity Index (INRI), that aims to assess the overall lability of brain dynamics without the use of extrinsic stimulation. The proposed technique uses three sigma EEG events as a trigger for ensuing changes to the directional derivative of signals across the EEG montage. RESULTS: The INRI had a positive relationship with GCS and was significantly different between various levels of consciousness. In comparison, classical band-limited power analysis did not show any specific patterns correlated to GCS. CONCLUSIONS: These findings suggest that reaching low variance EEG activation patterns becomes progressively harder as the level of consciousness of patients deteriorate, and provide a quantitative index based on passive measurements that characterize this change. SIGNIFICANCE: Our results emphasize the role of intrinsic brain dynamics in assessing the level of consciousness in coma patients and the possibility of employing simple electrophysiological measures to recognize the severity of disorders of consciousness (DOC).
Assuntos
Coma/diagnóstico , Estado de Consciência , Eletroencefalografia/métodos , Adulto , Idoso , Algoritmos , Encéfalo/fisiopatologia , Coma/classificação , Eletroencefalografia/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
Recent studies suggest that disruptions in resting state functional connectivity - a measure of stationary statistical association between brain regions - can be used as an objective marker of brain injury. However, fewer characterizations have examined the disruption of intrinsic brain dynamics after brain injury. Here, we examine this issue using electroencephalographic (EEG) data from brain-injured patients, together with a control analysis wherein we quantify the effect of the injury on the ability of intrinsic event responses to traverse their respective state spaces. More specifically, the lability of intrinsically evoked brain activity was assessed by collapsing three sigma event responses in all channels of the obtained EEG signals into a low-dimensional space. The directional derivative of these responses was then used to assay the extent to which brain activity reaches low-variance subspaces. Our findings suggest that intrinsic dynamics extracted from resting state EEG signals can differentiate various levels of consciousness in severe cases of coma. More specifically the cost of moving from one state to another in the state-space trajectories of the underlying dynamics becomes lower as the level of consciousness of patients deteriorates.