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Subconcussive head impacts are associated with the development of acute and chronic cognitive deficits. We recently reported that high-frequency head impact (HFHI) causes chronic cognitive deficits in mice through synaptic changes. To better understand the mechanisms underlying HFHI-induced memory decline, we used TRAP2/Ai32 transgenic mice to enable visualization and manipulation of memory engrams. We labeled the fear memory engram in male and female mice exposed to an aversive experience and subjected them to sham or HFHI. Upon subsequent exposure to natural memory recall cues, sham, but not HFHI, mice successfully retrieved fearful memories. In sham mice the hippocampal engram neurons exhibited synaptic plasticity, evident in amplified AMPA:NMDA ratio, enhanced AMPA-weighted tau, and increased dendritic spine volume compared with nonengram neurons. In contrast, although HFHI mice retained a comparable number of hippocampal engram neurons, these neurons did not undergo synaptic plasticity. This lack of plasticity coincided with impaired activation of the engram network, leading to retrograde amnesia in HFHI mice. We validated that the memory deficits induced by HFHI stem from synaptic plasticity impairments by artificially activating the engram using optogenetics and found that stimulated memory recall was identical in both sham and HFHI mice. Our work shows that chronic cognitive impairment after HFHI is a result of deficiencies in synaptic plasticity instead of a loss in neuronal infrastructure, and we can reinstate a forgotten memory in the amnestic brain by stimulating the memory engram. Targeting synaptic plasticity may have therapeutic potential for treating memory impairments caused by repeated head impacts.
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Amnésia , Memória , Masculino , Camundongos , Feminino , Animais , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Hipocampo/fisiologia , Camundongos TransgênicosRESUMO
Traumatic brain injury (TBI) is a major cause of disability and mortality worldwide, particularly among the elderly, yet our mechanistic understanding of what renders the post-traumatic brain vulnerable to poor outcomes, and susceptible to neurological disease, is incomplete. It is well established that dysregulated and sustained immune responses elicit negative consequences after TBI; however, our understanding of the neuroimmune interface that facilitates crosstalk between central and peripheral immune reservoirs is in its infancy. The meninges serve as the interface between the brain and the immune system, facilitating important bi-directional roles in both healthy and disease settings. It has been previously shown that disruption of this system exacerbates neuroinflammation in age-related neurodegenerative disorders such as Alzheimer's disease; however, we have an incomplete understanding of how the meningeal compartment influences immune responses after TBI. In this manuscript, we will offer a detailed overview of the holistic nature of neuroinflammatory responses in TBI, including hallmark features observed across clinical and animal models. We will highlight the structure and function of the meningeal lymphatic system, including its role in immuno-surveillance and immune responses within the meninges and the brain. We will provide a comprehensive update on our current knowledge of meningeal-derived responses across the spectrum of TBI, and identify new avenues for neuroimmune modulation within the neurotrauma field.
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Lesões Encefálicas Traumáticas , Meninges , Doenças Neuroinflamatórias , Lesões Encefálicas Traumáticas/imunologia , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/patologia , Humanos , Animais , Meninges/imunologia , Meninges/patologia , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/etiologia , Doenças Neuroinflamatórias/patologia , Neuroimunomodulação/fisiologia , Neuroimunomodulação/imunologiaRESUMO
The high-power, short-wavelength operation of a thulium-doped silica fiber laser at 1726 nm has been demonstrated in a core-pumped monolithic (all-fiber) resonator configuration, in-band pumped by a high-power erbium-only fiber laser operating at 1580 nm. The thulium fiber laser yielded 47 W in a single-spatial-mode output beam for 60-W absorbed pump power. The corresponding slope efficiency, with respect to an absorbed pump power of 80%, compares favorably with the theoretical maximum (Stokes) efficiency of 91.5%. The prospects for further scaling of single-mode power in this wavelength regime to >100 W are considered, as well as the potential applications for high-power lasers operating in this difficult-to-reach wavelength band.
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One challenge of integrating of passive, microparticles manipulation techniques into multifunctional microfluidic devices is coupling the continuous-flow format of most systems with the often batch-type operation of particle separation systems. Here, a passive fluidic technique-one-way particle transport-that can conduct microparticle operations in a closed fluidic circuit is presented. Exploiting pass/capture interactions between microparticles and asymmetric traps, this technique accomplishes a net displacement of particles in an oscillatory flow field. One-way particle transport is achieved through four kinds of trap-particle interactions: mechanical capture of the particle, asymmetric interactions between the trap and the particle, physical collision of the particle with an obstacle, and lateral shift of the particle into a particle-trapping stream. The critical dimensions for those four conditions are found by numerically solving analytical mass balance equations formulated using the characteristics of the flow field in periodic obstacle arrays. Visual observation of experimental trap-particle dynamics in low Reynolds number flow (<0.01) confirms the validity of the theoretical predictions. This technique can transport hundreds of microparticles across trap rows in only a few fluid oscillations (<500 ms per oscillation) and separate particles by their size differences.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Dispositivos Lab-On-A-Chip , Tamanho da PartículaRESUMO
The activation of resident microglial cells, alongside the infiltration of peripheral macrophages, are key neuroinflammatory responses to traumatic brain injury (TBI) that are directly associated with neuronal death. Sexual disparities in response to TBI have been previously reported; however it is unclear whether a sex difference exists in neuroinflammatory progression after TBI. We exposed male and female mice to moderate-to-severe controlled cortical impact injury and studied glial cell activation in the acute and chronic stages of TBI using immunofluorescence and in situ hybridization analysis. We found that the sex response was completely divergent up to 7 days postinjury. TBI caused a rapid and pronounced cortical microglia/macrophage activation in male mice with a prominent activated phenotype that produced both pro- (IL-1ß and TNFα) and anti-inflammatory (Arg1 and TGFß) cytokines with a single-phase, sustained peak from 1 to 7 days. In contrast, TBI caused a less robust microglia/macrophage phenotype in females with biphasic pro-inflammatory response peaks at 4 h and 7 days, and a delayed anti-inflammatory mRNA peak at 30 days. We further report that female mice were protected against acute cell loss after TBI, with male mice demonstrating enhanced astrogliosis, neuronal death, and increased lesion volume through 7 days post-TBI. Collectively, these findings indicate that TBI leads to a more aggressive neuroinflammatory profile in male compared with female mice during the acute and subacute phases postinjury. Understanding how sex affects the course of neuroinflammation following brain injury is a vital step toward developing personalized and effective treatments for TBI.
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Lesões Encefálicas Traumáticas/imunologia , Inflamação/fisiopatologia , Caracteres Sexuais , Animais , Arginase/metabolismo , Astrócitos/imunologia , Astrócitos/patologia , Lesões Encefálicas Traumáticas/patologia , Morte Celular , Modelos Animais de Doenças , Feminino , Gliose/imunologia , Gliose/patologia , Inflamação/patologia , Ativação de Macrófagos/imunologia , Ativação de Macrófagos/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Microglia/imunologia , Microglia/patologia , Neuroimunomodulação/fisiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Leakage of lead and other heavy metals into drinking water is a significant health risk and one that is not easily detected. We have developed simple sensors containing only platinum electrodes for the detection of heavy metal contamination in drinking water. The two-electrode sensor can identify the existence of a variety of heavy metals in drinking water, and the four-electrode sensor can distinguish lead from other heavy metals in solution. No false-positive response is generated when the sensors are placed in simulated and actual tap water contaminated by heavy metals. Lead detection on the four-electrode sensor is not affected by the presence of common ions in tap water. Experimental results suggest the sensors can be embedded in water service lines for long-time use until lead or other heavy metals are detected. With its low cost (â¼$0.10/sensor) and the possibility of long-term operation, the sensors are ideal for heavy metal detection of drinking water.
Assuntos
Água Potável/análise , Técnicas Eletroquímicas/instrumentação , Chumbo/análise , Poluentes Químicos da Água/análise , Impedância Elétrica , Técnicas Eletroquímicas/economia , Técnicas Eletroquímicas/métodos , Eletrodos , Platina/químicaRESUMO
Viscosity measurements have a wide range of applications from industrial chemical production to medical diagnosis. In this work, we have developed a simple droplet-based, water-in-oil continuous viscometer capable of measuring viscosity changes in 10 s or less and consuming a total sample volume of less than 1 µL/h. The viscometer employs a flow-focusing geometry and generates droplets under constant pressure. The length of the droplets (Ld) is highly correlated to the aqueous-phase viscosity (µaq) at high ratios of aqueous-inlet to oil-inlet pressure (AIP/OIP), yielding a linear relationship between µaq and 1/(Ld - Lc) where Lc is the minimal obtainable droplet length and approximately equals to the width of the droplet-generating channel. Theoretical analysis verifies this linear relationship, and the resulting equations can be used to optimize the design of the device such as the channel width, depth, and length. The viscometer can be used for Newtonian fluids and, by accurately calculating the shear rate, for non-Newtonian fluids such as Boger fluids and shear thinning fluids. In these latter cases, the shear rates depend on the velocity of the aqueous phase and can be adjusted by varying the input pressures. The applicable range of viscosity measurements depends on the oil-phase viscosity (µoil), and viscosities within the range of 0.01-10 µoil can be measured reliably with less than 5% error.
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BACKGROUND: NADPH oxidase (NOX2) is an enzyme system that generates reactive oxygen species (ROS) in microglia and macrophages. Excessive ROS production is linked with neuroinflammation and chronic neurodegeneration following traumatic brain injury (TBI). Redox signaling regulates macrophage/microglial phenotypic responses (pro-inflammatory versus anti-inflammatory), and NOX2 inhibition following moderate-to-severe TBI markedly reduces pro-inflammatory activation of macrophages/microglia resulting in concomitant increases in anti-inflammatory responses. Here, we report the signaling pathways that regulate NOX2-dependent macrophage/microglial phenotype switching in the TBI brain. METHODS: Bone marrow-derived macrophages (BMDMs) prepared from wildtype (C57Bl/6) and NOX2 deficient (NOX2-/-) mice were treated with lipopolysaccharide (LPS; 10 ng/ml), interleukin-4 (IL-4; 10 ng/ml), or combined LPS/IL-4 to investigate signal transduction pathways associated with macrophage activation using western immunoblotting and qPCR analyses. Signaling pathways and activation markers were evaluated in ipsilateral cortical tissue obtained from adult male wildtype and NOX2-/- mice that received moderate-level controlled cortical impact (CCI). A neutralizing anti-IL-10 approach was used to determine the effects of IL-10 on NOX2-dependent transitions from pro- to anti-inflammatory activation states. RESULTS: Using an LPS/IL-4-stimulated BMDM model that mimics the mixed pro- and anti-inflammatory responses observed in the injured cortex, we show that NOX2-/- significantly reduces STAT1 signaling and markers of pro-inflammatory activation. In addition, NOX2-/- BMDMs significantly increase anti-inflammatory marker expression; IL-10-mediated STAT3 signaling, but not STAT6 signaling, appears to be critical in regulating this anti-inflammatory response. Following moderate-level CCI, IL-10 is significantly increased in microglia/macrophages in the injured cortex of NOX2-/- mice. These changes are associated with increased STAT3 activation, but not STAT6 activation, and a robust anti-inflammatory response. Neutralization of IL-10 in NOX2-/- BMDMs or CCI mice blocks STAT3 activation and the anti-inflammatory response, thereby demonstrating a critical role for IL-10 in regulating NOX2-dependent transitions between pro- and anti-inflammatory activation states. CONCLUSIONS: These studies indicate that following TBI NOX2 inhibition promotes a robust anti-inflammatory response in macrophages/microglia that is mediated by the IL-10/STAT3 signaling pathway. Thus, therapeutic interventions that inhibit macrophage/microglial NOX2 activity may improve TBI outcomes by not only limiting pro-inflammatory neurotoxic responses, but also enhancing IL-10-mediated anti-inflammatory responses that are neuroprotective.
Assuntos
Lesões Encefálicas Traumáticas/imunologia , Inflamação/patologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , NADPH Oxidase 2/deficiência , Animais , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismoRESUMO
Mild traumatic brain injury (mTBI) is an emerging risk for chronic behavioral, cognitive, and neurodegenerative conditions. Athletes absorb several hundred mTBIs each year; however, rodent models of repeat mTBI (rmTBI) are often limited to impacts in the single digits. Herein, we describe the effects of 30 rmTBIs, examining structural and pathological changes in mice up to 365 days after injury. We found that single mTBI causes a brief loss of consciousness and a transient reduction in dendritic spines, reflecting a loss of excitatory synapses. Single mTBI does not cause axonal injury, neuroinflammation, or cell death in the gray or white matter. Thirty rmTBIs with a 1-day interval between each mTBI do not cause dendritic spine loss; however, when the interinjury interval is increased to 7 days, dendritic spine loss is reinstated. Thirty rmTBIs cause white matter pathology characterized by positive silver and Fluoro-Jade B staining, and microglial proliferation and activation. This pathology continues to develop through 60 days, and is still apparent at 365 days, after injury. However, rmTBIs did not increase ß-amyloid levels or tau phosphorylation in the 3xTg-AD mouse model of Alzheimer disease. Our data reveal that single mTBI causes a transient loss of synapses, but that rmTBIs habituate to repetitive injury within a short time period. rmTBI causes the development of progressive white matter pathology that continues for months after the final impact.
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Lesões Encefálicas/patologia , Espinhas Dendríticas/patologia , Substância Branca/patologia , Amiloide/metabolismo , Animais , Comportamento Animal , Concussão Encefálica/patologia , Lesões Encefálicas/metabolismo , Traumatismos Craniocerebrais/metabolismo , Traumatismos Craniocerebrais/patologia , Espinhas Dendríticas/metabolismo , Modelos Animais de Doenças , Fluoresceínas , Complexo de Golgi , Humanos , Inflamação , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Recidiva , Inconsciência , Substância Branca/metabolismo , Proteínas tau/metabolismoRESUMO
Monitoring of the pH, oxidation-reduction-potential (ORP), and conductivity of aqueous samples is typically performed using multiple sensors. To minimize the size and cost of these sensors for practical applications, we have investigated the use of a single sensor constructed with only bare platinum electrodes deposited on a glass substrate. The sensor can measure pH from 4 to 10 while simultaneously measuring ORP from 150 to 800 mV. The device can also measure conductivity up to 8000 µS/cm in the range of 10 °C to 50 °C, and all these measurements can be made even if the water samples contain common ions found in residential water. The sensor is inexpensive (i.e., ~$0.10/unit) and has a sensing area below 1 mm², suggesting that the unit is cost-efficient, robust, and widely applicable, including in microfluidic systems.
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The trans-activating response of DNA/RNA-binding protein (TDP)-43 pathology is associated with many neurodegenerative diseases via unknown mechanisms. Here, we use a transgenic mouse model over-expressing human wild-type neuronal TDP-43 to study the effects of TDP-43 pathology on glutamate metabolism and synaptic function. We found that neuronal TDP-43 over-expression affects synaptic protein expression, including Synapsin I, and alters surrounding astrocytic function. TDP-43 over-expression is associated with an increase in glutamate and γ-amino butyric acid and reduction of glutamine and aspartate levels, indicating impairment of presynaptic terminal. TDP-43 also decreases tricarboxylic acid cycle metabolism and induces oxidative stress via lactate accumulation. Neuronal TDP-43 does not alter microglia activity or significantly changes systemic and brain inflammatory markers compared to control. We previously demonstrated that brain-penetrant tyrosine kinase inhibitors (TKIs), nilotinib and bosutinib, reduce TDP-43-induced cell death in transgenic mice. Here, we show that TKIs reverse the effects of TDP-43 on synaptic proteins, increase astrocytic function and restore glutamate and neurotransmitter balance in TDP-43 mice. Nilotinib, but not bosutinib, reverses mitochondrial impairment and oxidative metabolism. Taken together, these data suggest that TKIs can attenuate TDP-43 toxicity and improve synaptic and astrocytic function, independent of microglial or other inflammatory effects. In conclusion, our data demonstrate novel mechanisms of the effects of neuronal TDP-43 over-expression on synaptic protein expression and alteration of astrocytic function.
Assuntos
Astrócitos/fisiologia , Proteínas de Ligação a DNA/biossíntese , Homeostase/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Sinapsinas/biossíntese , Animais , Astrócitos/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Homeostase/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapsinas/genéticaRESUMO
The apolipoprotein E4 (APOE-ε4) allele is the strongest genetic risk factor for developing late-onset Alzheimer's disease, and may predispose individuals to Alzheimer's-related cognitive decline by affecting normal brain function early in life. To investigate the impact of human APOE alleles on cognitive performance in mice, we trained 3-mo-old APOE targeted replacement mice (E2, E3, and E4) in the Barnes maze to locate and enter a target hole along the perimeter of the maze. Long-term spatial memory was probed 24 h and 72 h after training. We found that young E4 mice exhibited significantly impaired spatial learning and memory in the Barnes maze compared to E3 mice. Deficits in spatial cognition were also present in a second independent cohort of E4 mice tested at 18 mo of age. In contrast, cognitive performance in the hidden platform water maze was not as strongly affected by APOE genotype. We also examined the dendritic morphology of neurons in the medial entorhinal cortex of 3-mo-old TR mice, neurons important to spatial learning functions. We found significantly shorter dendrites and lower spine densities in basal shaft dendrites of E4 mice compared to E3 mice, consistent with spatial learning and memory deficits in E4 animals. These findings suggest that human APOE-ε4 may affect cognitive function and neuronal morphology early in life.
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Apolipoproteína E4/genética , Espinhas Dendríticas/metabolismo , Córtex Entorrinal/metabolismo , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Neurônios/metabolismo , Animais , Apolipoproteína E4/metabolismo , Comportamento Animal , Dendritos/genética , Dendritos/metabolismo , Espinhas Dendríticas/genética , Camundongos , Camundongos Transgênicos , Comportamento Espacial/fisiologiaRESUMO
Background: Preventing childhood obesity and associated comorbidities is often hampered by disproportionate disparity in healthcare provision in minority ethnic populations. This study contextualized factors influencing childhood obesity and related comorbidity from the perspectives and experiences of parents of ethnic minority populations. Methods: Following ethical approval, families (n = 180) from ethnic minority populations in the Northeast of England were contacted through flyers, community social groups and online forum. Of the 180 families contacted, 22 expressed interests, of whom 12 parents were eligible to participate in the study, and one family dropped out due to time constraints. Therefore 11 parents from ethnic minority communities living with at least one child with obesity were interviewed. Each family was separately visited at home and took part in a semi-structured interview based on the study's qualitative, descriptive phenomenological design. Nine of the families had one child who was diagnosed with an obesity-related comorbidity (non-alcoholic fatty liver disease, musculoskeletal problems or respiratory disorder). Semi-structured interviews were standardized around parents' perspective and experience on how their children were impacted by obesity and comorbidities, healthcare preventative interventions including lifestyle physical activity and nutrition, and views on tackling obesity impact on their lives. All interviews were analyzed using qualitative thematic analysis. Results: Parents' perspectives revealed 11 themes centered around experience of living with a child with obesity, risks, and impact of obesity related Non-Communicable Diseases; and access to support, and barriers unique to minority ethnic groups. Parents revealed social disadvantages, fear of victimization by social services, perceptions on their cultural and religious traditions, and racial stigmatization related to their child's weight. Parents reported closer bonding with their children to protect them from the untoward consequences of overweight, and little awareness of healthcare obesity prevention programs. Work pressure, lack of time, absence of guidance from professionals were seen as barriers to healthy lifestyle, while support from friends and closer family bond in adopting healthy lifestyle behaviors were facilitators. However, there was little awareness or access to current healthcare obesity preventive offerings. Conclusion: Minority ethnic communities' perspective on childhood obesity prevention does not match the healthcare system preventative offerings. Community and family-oriented obesity preventative approaches, especially lifestyle interventions are needed beyond those administered by the primary healthcare system.
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Comorbidade , Pais , Obesidade Infantil , Pesquisa Qualitativa , Humanos , Inglaterra , Obesidade Infantil/psicologia , Obesidade Infantil/etnologia , Feminino , Masculino , Pais/psicologia , Criança , Adulto , População Negra/estatística & dados numéricos , População Negra/psicologia , Povo Asiático/psicologia , Povo Asiático/estatística & dados numéricos , Grupos Minoritários/estatística & dados numéricos , Grupos Minoritários/psicologia , Minorias Étnicas e Raciais/estatística & dados numéricos , Pré-Escolar , Entrevistas como Assunto , Pessoa de Meia-Idade , Etnicidade/psicologia , Etnicidade/estatística & dados numéricosRESUMO
BACKGROUND: Locally advanced, bulky, unresectable sarcomas cause significant tumour mass effects, leading to burdensome symptoms. We have developed a novel Partially Ablative Body Radiotherapy (PABR) technique that delivers a high, ablative dose to the tumour core and a low, palliative dose to its periphery aiming to increase overall tumour response without significantly increasing treatment toxicity. AIM: This study aims to report the safety and oncologic outcomes of PABR in patients with bulky, unresectable sarcomas. METHODS AND MATERIALS: A total of 18 patients with histologically proven sarcoma treated with PABR from January 2020 to October 2023 were retrospectively reviewed. The primary endpoints were symptomatic and structural response rates. Secondary endpoints were overall survival, freedom from local progression, freedom from distant progression, and acute and late toxicity rates. RESULTS: All patients had tumours ≥5 cm with a median tumour volume of 985 cc, and the most common symptom was pain. The median age is 72.5 years and 44.5 % were ECOG 2-3. The most common regimen used was 20 Gy in 5 fractions with an intratumoral boost dose of 50 Gy (83.3 %). After a median follow-up of 11 months, 88.9 % of patients exhibited a partial response with a mean absolute tumour volume reduction of 49.5 %. All symptomatic patients experienced symptom improvement. One-year OS, FFLP and FFDP were 61 %, 83.3 % and 34.8 %, respectively. There were no grade 3 or higher toxicities. CONCLUSION: PABR for bulky, unresectable sarcomas appears to be safe and may provide good symptomatic response, tumour debulking, and local control. Further study is underway.
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Cuidados Paliativos , Sarcoma , Humanos , Sarcoma/radioterapia , Sarcoma/patologia , Sarcoma/cirurgia , Sarcoma/mortalidade , Masculino , Cuidados Paliativos/métodos , Feminino , Idoso , Estudos Retrospectivos , Pessoa de Meia-Idade , Idoso de 80 Anos ou mais , Carga Tumoral , Adulto , Dosagem RadioterapêuticaRESUMO
Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in many forms of cancer including NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested in NB. We propose that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501 will result in enhanced NB growth inhibition. Single and combination drug treatments were conducted on three NB cell lines. DFMO IC50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 µM in NB. The combination treatment resulted in hypophosphorylation of retinoblastoma protein (Rb), suggesting growth inhibition via G1 cell cycle arrest. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hr suggested apoptosis. The combination treatment depleted intracellular polyamine pools and decreased intracellular ATP, further verifying growth inhibition. Given the current lack of effective therapies for patients with relapsed/refractory NB and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB.
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Poliaminas Biogênicas/metabolismo , Proliferação de Células/efeitos dos fármacos , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Neuroblastoma/tratamento farmacológico , Inibidores da Ornitina Descarboxilase , Transporte Biológico/efeitos dos fármacos , Caspase 3/fisiologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Genes myc , Humanos , Neuroblastoma/patologiaRESUMO
Traumatic Brain Injury (TBI) is a major cause of disability and mortality, particularly among the elderly, yet our mechanistic understanding of how age renders the post-traumatic brain vulnerable to poor clinical outcomes and susceptible to neurological disease remains poorly understood. It is well established that dysregulated and sustained immune responses contribute to negative outcomes after TBI, however our understanding of the interactions between central and peripheral immune reservoirs is still unclear. The meninges serve as the interface between the brain and the immune system, facilitating important bi-directional roles in healthy and disease settings. It has been previously shown that disruption of this system exacerbates inflammation in age related neurodegenerative disorders such as Alzheimer's disease, however we have an incomplete understanding of how the meningeal compartment influences immune responses after TBI. Here, we examine the meningeal tissue and its response to brain injury in young (3-months) and aged (18-months) mice. Utilizing a bioinformatic approach, high-throughput RNA sequencing demonstrates alterations in the meningeal transcriptome at sub-acute (7-days) and chronic (1 month) timepoints after injury. We find that age alone chronically exacerbates immunoglobulin production and B cell responses. After TBI, adaptive immune response genes are up-regulated in a temporal manner, with genes involved in T cell responses elevated sub-acutely, followed by increases in B cell related genes at chronic time points after injury. Pro-inflammatory cytokines are also implicated as contributing to the immune response in the meninges, with ingenuity pathway analysis identifying interferons as master regulators in aged mice compared to young mice following TBI. Collectively these data demonstrate the temporal series of meningeal specific signatures, providing insights into how age leads to worse neuroinflammatory outcomes in TBI.
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Traumatic brain injury (TBI) and repetitive head impacts can result in a wide range of neurological symptoms. Despite being the most common neurological disorder in the world, repeat head impacts and TBI do not have any FDA-approved treatments. Single neuron modeling allows researchers to extrapolate cellular changes in individual neurons based on experimental data. We recently characterized a model of high frequency head impact (HFHI) with a phenotype of cognitive deficits associated with decreases in neuronal excitability of CA1 neurons and synaptic changes. While the synaptic changes have been interrogated in vivo, the cause and potential therapeutic targets of hypoexcitability following repetitive head impacts are unknown. Here, we generated in silico models of CA1 pyramidal neurons from current clamp data of control mice and mice that sustained HFHI. We use a directed evolution algorithm with a crowding penalty to generate a large and unbiased population of plausible models for each group that approximated the experimental features. The HFHI neuron model population showed decreased voltage gated sodium conductance and a general increase in potassium channel conductance. We used partial least squares regression analysis to identify combinations of channels that may account for CA1 hypoexcitability after HFHI. The hypoexcitability phenotype in models was linked to A- and M-type potassium channels in combination, but not by any single channel correlations. We provide an open access set of CA1 pyramidal neuron models for both control and HFHI conditions that can be used to predict the effects of pharmacological interventions in TBI models.
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Neurônios , Canais de Potássio , Camundongos , Animais , Canais de Potássio/farmacologia , Potenciais de Ação/fisiologia , Neurônios/fisiologia , Células Piramidais/fisiologiaRESUMO
The pathophysiological changes that occur after traumatic brain injury (TBI) can lead to the development of post-traumatic epilepsy, a life-long complication of brain trauma. The etiology of post-traumatic epilepsy remains unknown, but TBI brains exhibit an abnormal excitatory / inhibitory balance. In this study, we examine how brain injury alters susceptibility to chemically-induced seizures in C57Bl/6J mice, and if pharmacological enhancement of glutamate transporters can reduce chronic post-traumatic seizures. We found that controlled cortical impact (CCI) mice display delayed susceptibility to pentylenetetrazol (PTZ)-induced seizures. While CCI mice have no change in seizure susceptibility at 7d post-injury (dpi), at 70dpi they have reduced latency to PTZ-induced seizure onset, higher seizure frequency and longer seizure duration. Quantification of glutamate transporter mRNA showed that levels of Scl1a2 and Scl1a3 mRNA were increased at 7dpi, but significantly decreased at 70dpi. To test if increased levels of glutamate transporters can ameliorate delayed-onset seizure susceptibility in TBI mice, we exposed a new cohort of mice to CCI and administered ceftriaxone (200mg/kg/day) for 14d from 55-70dpi. We found that ceftriaxone significantly increased Scl1a2 and Scl1a3 in CCI mouse brain at 70dpi, and prevented the susceptibility of CCI mice to PTZ-induced seizures. This study demonstrates cortical impact can induce a delayed-onset seizure phenotype in mice. Delayed (55dpi) ceftriaxone treatment enhances glutamate transporter mRNA in the CCI brain, and reduces PTZ-induced seizures in CCI mice.
Assuntos
Lesões Encefálicas Traumáticas , Epilepsia Pós-Traumática , Humanos , Camundongos , Animais , Ceftriaxona/farmacologia , Ceftriaxona/uso terapêutico , Tempo para o Tratamento , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/tratamento farmacológico , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Convulsões/complicações , Pentilenotetrazol/toxicidade , Camundongos Endogâmicos C57BL , Glutamatos , Modelos Animais de DoençasRESUMO
Urinary chloride concentration is a valuable health metric that can aid in the early detection of serious conditions, such as acid base disorders, acute heart failure, and incidences of acute renal failure in the intensive care unit. Physiologically, urinary chloride levels frequently change and are difficult to measure, involving time-consuming and inconvenient lab testing. Thus, near real-time simple sensors are needed to quickly provide actionable data to inform diagnostic and treatment decisions that affect health outcomes. Here, we introduce a chronopotentiometric sensor that utilizes commercially available screen-printed electrodes to accurately quantify clinically relevant chloride concentrations (5-250 mM) in seconds, with no added reagents or electrode surface modification. Initially, the sensor's performance was optimized through the proper selection of current density at a specific chloride concentration, using electrical response data in conjunction with scanning electron microscopy. We developed a unique swept current density algorithm to resolve the entire clinically relevant chloride concentration range, and the chloride sensors can be reliably reused for chloride concentrations less than 50 mM. Lastly, we explored the impact of pH, temperature, conductivity, and additional ions (i.e., artificial urine) on the sensor signal, in order to determine sensor feasibility in complex biological samples. This study provides a path for further development of a portable, near real-time sensor for the quantification of urinary chloride.
Assuntos
Cloretos , Técnicas Eletroquímicas , Eletrodos , Microscopia Eletrônica de VarreduraRESUMO
Background: Surgery and/or platinum-based chemoradiation remain standard of care for patients with head and neck squamous cell carcinoma (HNSCC). While these therapies are effective in a subset of patients, a substantial proportion experience recurrence or treatment resistance. As cisplatin mediates cytotoxicity through oxidative stress while polyamines play a role in redox regulation, we posited that combining cisplatin with polyamine transport inhibitor, AMXT-1501, would increase oxidative stress and tumor cell death in HNSCC cells. Methods: Cell proliferation was measured in syngeneic mouse HNSCC cell lines treated with cisplatin ± AMXT-1501. Synergy was determined by administering cisplatin and AMXT-1501 at a ratio of 1:10 to cancer cells in vitro . Cancer cells were transferred onto mouse flanks to test the efficacy of treatments in vivo . Reactive oxygen species (ROS) were measured. Cellular apoptosis was measured with flow cytometry using Annexin V/PI staining. High-performance liquid chromatography (HPLC) was used to quantify polyamines in cell lines. Cell viability and ROS were measured in the presence of exogenous cationic amino acids. Results: The combination of cisplatin and AMXT-1501 synergize in vitro on HNSCC cell lines. In vivo combination treatment resulted in tumor growth inhibition greater than either treatment individually. The combination treatment increased ROS production and induced apoptotic cell death. HPLC revealed the synergistic mechanism was independent of intracellular polyamine levels. Supplementation of cationic amino acids partially rescued cancer cell viability and reduced ROS. Conclusion: AMXT-1501 enhances the cytotoxic effects of cisplatin in vitro and in vivo in aggressive HNSCC cell lines through a polyamine-independent mechanism.