RESUMO
Obesity increases the morbidity and mortality of traumatic brain injury (TBI). Detailed analyses of transcriptomic changes in the brain and adipose tissue were performed to elucidate the interactive effects between high-fat diet-induced obesity (DIO) and TBI. Adult male mice were fed a high-fat diet (HFD) for 12 weeks prior to experimental TBI and continuing after injury. High-throughput transcriptomic analysis using Nanostring panels of the total visceral adipose tissue (VAT) and cellular components in the brain, followed by unsupervised clustering, principal component analysis, and IPA pathway analysis were used to determine shifts in gene expression patterns and molecular pathway activity. Cellular populations in the cortex and hippocampus, as well as in VAT, during the chronic phase after combined TBI-HFD showed amplification of central and peripheral microglia/macrophage responses, including superadditive changes in selected gene expression signatures and pathways. Furthermore, combined TBI and HFD caused additive dysfunction in Y-Maze, Novel Object Recognition (NOR), and Morris water maze (MWM) cognitive function tests. These novel data suggest that HFD-induced obesity and TBI can independently prime and support the development of altered states in brain microglia and VAT, including the disease-associated microglia/macrophage (DAM) phenotype observed in neurodegenerative disorders. The interaction between HFD and TBI promotes a shift toward chronic reactive microglia/macrophage transcriptomic signatures and associated pro-inflammatory disease-altered states that may, in part, underlie the exacerbation of cognitive deficits. Thus, targeting of HFD-induced reactive cellular phenotypes, including in peripheral adipose tissue immune cell populations, may serve to reduce microglial maladaptive states after TBI, attenuating post-traumatic neurodegeneration and neurological dysfunction.
Assuntos
Lesões Encefálicas Traumáticas , Encéfalo , Disfunção Cognitiva , Dieta Hiperlipídica , Macrófagos , Camundongos Endogâmicos C57BL , Microglia , Animais , Dieta Hiperlipídica/efeitos adversos , Microglia/metabolismo , Microglia/patologia , Masculino , Camundongos , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/patologia , Disfunção Cognitiva/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/metabolismo , Encéfalo/patologia , Encéfalo/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Reconhecimento Psicológico/fisiologia , Obesidade/patologia , Obesidade/complicações , Aprendizagem em Labirinto/fisiologiaRESUMO
BACKGROUND: It is well established that traumatic brain injury (TBI) causes acute and chronic alterations in systemic immune function and that systemic immune changes contribute to posttraumatic neuroinflammation and neurodegeneration. However, how TBI affects bone marrow (BM) hematopoietic stem/progenitor cells chronically and to what extent such changes may negatively impact innate immunity and neurological function has not been examined. METHODS: To further understand the role of BM cell derivatives on TBI outcome, we generated BM chimeric mice by transplanting BM from chronically injured or sham (i.e., 90 days post-surgery) congenic donor mice into otherwise healthy, age-matched, irradiated CD45.2 C57BL/6 (WT) hosts. Immune changes were evaluated by flow cytometry, multiplex ELISA, and NanoString technology. Moderate-to-severe TBI was induced by controlled cortical impact injury and neurological function was measured using a battery of behavioral tests. RESULTS: TBI induced chronic alterations in the transcriptome of BM lineage-c-Kit+Sca1+ (LSK+) cells in C57BL/6 mice, including modified epigenetic and senescence pathways. After 8 weeks of reconstitution, peripheral myeloid cells from TBIâWT mice showed significantly higher oxidative stress levels and reduced phagocytic activity. At eight months after reconstitution, TBIâWT chimeric mice were leukopenic, with continued alterations in phagocytosis and oxidative stress responses, as well as persistent neurological deficits. Gene expression analysis revealed BM-driven changes in neuroinflammation and neuropathology after 8 weeks and 8 months of reconstitution, respectively. Chimeric mice subjected to TBI at 8 weeks and 8 months post-reconstitution showed that longer reconstitution periods (i.e., time post-injury) were associated with increased microgliosis and leukocyte infiltration. Pre-treatment with a senolytic agent, ABT-263, significantly improved behavioral performance of aged C57BL/6 mice at baseline, although it did not attenuate neuroinflammation in the acutely injured brain. CONCLUSIONS: TBI causes chronic activation and progressive dysfunction of the BM stem/progenitor cell pool, which drives long-term deficits in hematopoiesis, innate immunity, and neurological function, as well as altered sensitivity to subsequent brain injury.
Assuntos
Lesões Encefálicas Traumáticas , Lesões Encefálicas , Camundongos , Animais , Doenças Neuroinflamatórias , Camundongos Endogâmicos C57BL , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas/patologia , Encéfalo/metabolismoRESUMO
The National Institutes of Health (NIH) recognizes that, despite HIV scientific advances, stigma and discrimination continue to be critical barriers to the uptake of evidence-based HIV interventions. Achieving the Ending the HIV Epidemic: A Plan for America (EHE) goals will require eliminating HIV-related stigma. NIH has a significant history of supporting HIV stigma research across its Institutes, Centers, and Offices (ICOs) as a research priority. This article provides an overview of NIH HIV stigma research efforts. Each ICO articulates how their mission shapes their interest in HIV stigma research and provides a summary of ICO-relevant scientific findings. Research gaps and/or future opportunities are identified throughout, with key research themes and approaches noted. Taken together, the collective actions on the part of the NIH, in tandem with a whole of government and whole of society approach, will contribute to achieving EHE's milestones.
RESUMEN: Los Institutos de Salud Nacional (NIH, siglas en inglés) reconocen que, a pesar de los avances en la prevención y el tratamiento, el estigma y la discriminación continúan siendo barreras críticas a la adopción de la prevención y el cuido basados en la evidencia. Las metas de Logrando el Fin de la Epidemia de VIH: Plan para América (EHE, siglas en inglés) requerirán la eliminación del estigma relacionado al VIH. Los NIH tienen una historia significativa apoyando la investigación del estigma relacionado al VIH a través de sus Institutos, Centros, y Oficinas (ICOs, siglas en inglés). Esta investigación es una prioridad fundamental y entrelazada para los ICOs. En este artículo, los autores de los NIH proveen una reseña sobre la investigación del estigma relacionado al VIH a través de los ICOs selectos. Cada ICO articula como su misión y prioridad dan forma a su interés en la investigación del estigma al VIH y provee una breve reseña de los hallazgos científicos pertinentes al ICO. Lagunas en la investigación relacionada a la misión, prioridades, y/o áreas de investigación futuras se identifican a través del artículo. También se apuntan en el resumen los temas de investigación claves y sus estrategias. En conjunto, las acciones colectivas de parte de los NIH, junto a la estrategia necesaria de parte del gobierno en su totalidad y de la sociedad en su totalidad, contribuirán al logro de las metas del EHE.
Assuntos
Infecções por HIV , Infecções por HIV/prevenção & controle , Humanos , National Institutes of Health (U.S.) , Estigma Social , Estados UnidosRESUMO
DNA damage and type I interferons (IFNs) contribute to inflammatory responses after traumatic brain injury (TBI). TBI-induced activation of microglia and peripherally-derived inflammatory macrophages may lead to tissue damage and neurological deficits. Here, we investigated the role of IFN-ß in secondary injury after TBI using a controlled cortical impact model in adult male IFN-ß-deficient (IFN-ß-/-) mice and assessed post-traumatic neuroinflammatory responses, neuropathology, and long-term functional recovery. TBI increased expression of DNA sensors cyclic GMP-AMP synthase and stimulator of interferon genes in wild-type (WT) mice. IFN-ß and other IFN-related and neuroinflammatory genes were also upregulated early and persistently after TBI. TBI increased expression of proinflammatory mediators in the cortex and hippocampus of WT mice, whereas levels were mitigated in IFN-ß-/- mice. Moreover, long-term microglia activation, motor, and cognitive function impairments were decreased in IFN-ß-/- TBI mice compared with their injured WT counterparts; improved neurological recovery was associated with reduced lesion volume and hippocampal neurodegeneration in IFN-ß-/- mice. Continuous central administration of a neutralizing antibody to the IFN-α/ß receptor (IFNAR) for 3 d, beginning 30 min post-injury, reversed early cognitive impairments in TBI mice and led to transient improvements in motor function. However, anti-IFNAR treatment did not improve long-term functional recovery or decrease TBI neuropathology at 28 d post-injury. In summary, TBI induces a robust neuroinflammatory response that is associated with increased expression of IFN-ß and other IFN-related genes. Inhibition of IFN-ß reduces post-traumatic neuroinflammation and neurodegeneration, resulting in improved neurological recovery. Thus, IFN-ß may be a potential therapeutic target for TBI.SIGNIFICANCE STATEMENT TBI frequently causes long-term neurological and psychiatric changes in head injury patients. TBI-induced secondary injury processes including persistent neuroinflammation evolve over time and can contribute to chronic neurological impairments. The present study demonstrates that TBI is followed by robust activation of type I IFN pathways, which have been implicated in microglial-associated neuroinflammation and chronic neurodegeneration. We examined the effects of genetic or pharmacological inhibition of IFN-ß, a key component of type I IFN mechanisms to address its role in TBI pathophysiology. Inhibition of IFN-ß signaling resulted in reduced neuroinflammation, attenuated neurobehavioral deficits, and limited tissue loss long after TBI. These preclinical findings suggest that IFN-ß may be a potential therapeutic target for TBI.
Assuntos
Dano Encefálico Crônico/fisiopatologia , Lesões Encefálicas Traumáticas/fisiopatologia , Interferon beta/fisiologia , Degeneração Neural/etiologia , Animais , Dano Encefálico Crônico/etiologia , Lesões Encefálicas Traumáticas/complicações , Córtex Cerebral/metabolismo , Comportamento Exploratório/fisiologia , Regulação da Expressão Gênica , Hipocampo/metabolismo , Inflamação , Interferon beta/biossíntese , Interferon beta/deficiência , Interferon beta/genética , Masculino , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/etiologia , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/fisiologia , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/fisiopatologia , Distribuição Aleatória , Receptor de Interferon alfa e beta/imunologia , Transdução de Sinais , Regulação para CimaRESUMO
Chronic neuroinflammation with sustained microglial activation occurs following severe traumatic brain injury (TBI) and is believed to contribute to subsequent neurodegeneration and neurological deficits. Microglia, the primary innate immune cells in brain, are dependent on colony stimulating factor 1 receptor (CSF1R) signaling for their survival. In this preclinical study, we examined the effects of delayed depletion of chronically activated microglia on functional recovery and neurodegeneration up to 3 months postinjury. A CSF1R inhibitor, Plexxikon (PLX) 5622, was administered to adult male C57BL/6J mice at 1 month after controlled cortical impact to remove chronically activated microglia, and the inhibitor was withdrawn 1-week later to allow for microglial repopulation. Following TBI, the repopulated microglia displayed a ramified morphology similar to that of Sham uninjured mice, whereas microglia in vehicle-treated TBI mice showed the typical chronic posttraumatic hypertrophic morphology. PLX5622 treatment limited TBI-associated neuropathological changes at 3 months postinjury; these included a smaller cortical lesion, reduced hippocampal neuron cell death, and decreased NOX2- and NLRP3 inflammasome-associated neuroinflammation. Furthermore, delayed depletion of chronically activated microglia after TBI led to widespread changes in the cortical transcriptome and altered gene pathways involved in neuroinflammation, oxidative stress, and neuroplasticity. Using a variety of complementary neurobehavioral tests, PLX5622-treated TBI mice also had improved long-term motor and cognitive function recovery through 3 months postinjury. Together, these studies demonstrate that chronic phase removal of neurotoxic microglia after TBI using CSF1R inhibitors markedly reduce chronic neuroinflammation and associated neurodegeneration, as well as related motor and cognitive deficits.SIGNIFICANCE STATEMENT Traumatic brain injury (TBI) is a debilitating neurological disorder that can seriously impact the patient's quality of life. Microglial-mediated neuroinflammation is induced after severe TBI and contributes to neurological deficits and on-going neurodegenerative processes. Here, we investigated the effect of breaking the neurotoxic neuroinflammatory loop at 1-month after controlled cortical impact in mice by pharmacological removal of chronically activated microglia using a colony stimulating factor 1 receptor (CSF1R) inhibitor, Plexxikon 5622. Overall, we show that short-term elimination of microglia during the chronic phase of TBI followed by repopulation results in long-term improvements in neurological function, suppression of neuroinflammatory and oxidative stress pathways, and a reduction in persistent neurodegenerative processes. These studies are clinically relevant and support new concepts that the therapeutic window for TBI may be far longer than traditionally believed if chronic and evolving microglial-mediated neuroinflammation can be inhibited or regulated in a precise manner.
Assuntos
Lesões Encefálicas Traumáticas/patologia , Microglia/efeitos dos fármacos , Degeneração Neural/patologia , Fármacos Neuroprotetores/farmacologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Animais , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/fisiopatologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologiaRESUMO
We have previously shown that treatment with a mGluR5 positive allosteric modulator (PAM) is neuroprotective after experimental traumatic brain injury (TBI), limiting post-traumatic neuroinflammation by reducing pro-inflammatory microglial activation and promoting anti-inflammatory and neuroprotective responses. However, the specific molecular mechanisms governing this anti-inflammatory shift in microglia remain unknown. Here we show that the mGluR5 PAM, VU0360172 (VuPAM), regulates microglial inflammatory responses through activation of Akt, resulting in the inhibition of GSK-3ß. GSK-3ß regulates the phosphorylation of CREB, thereby controlling the expression of inflammation-related genes and microglial plasticity. The anti-inflammatory action of VuPAM in microglia is reversed by inhibiting Akt/GSK-3ß/CREB signaling. Using a well-characterized TBI model and CX3CR1gfp/+ mice to visualize microglia in vivo, we demonstrate that VuPAM enhances Akt/GSK-3ß/CREB signaling in the injured cortex, as well as anti-inflammatory microglial markers. Furthermore, in situ analysis revealed that GFP + microglia in the cortex of VuPAM-treated TBI mice co-express pCREB and the anti-inflammatory microglial phenotype marker YM1. Taken together, our data show that VuPAM decreases pro-inflammatory microglial activation by modulating Akt/GSK-3ß/CREB signaling. These findings serve to clarify the potential neuroprotective mechanisms of mGluR5 PAM treatment after TBI, and suggest novel therapeutic targets for post-traumatic neuroinflammation. Cover Image for this issue: https://doi.org/10.1111/jnc.15048.
Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Niacinamida/análogos & derivados , Receptor de Glutamato Metabotrópico 5/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Masculino , Camundongos , Microglia/metabolismo , Niacinamida/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Glutamato Metabotrópico 5/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Extracellular vesicles (EVs) have been implicated mechanistically in the pathobiology of neurodegenerative disorders, including central nervous system injury. However, the role of EVs in spinal cord injury (SCI) has received limited attention to date. Moreover, technical limitations related to EV isolation and characterization methods can lead to misleading or contradictory findings. Here, we examined changes in plasma EVs after mouse SCI at multiple timepoints (1d, 3d, 7d, 14d) using complementary measurement techniques. Plasma EVs isolated by ultracentrifugation (UC) were decreased at 1d post-injury, as shown by nanoparticle tracking analysis (NTA), and paralleled an overall reduction in total plasma extracellular nanoparticles. Western blot (WB) analysis of UC-derived plasma EVs revealed increased expression of the tetraspanin exosome marker, CD81, between 1d and 7d post-injury. To substantiate these findings, we performed interferometric and fluorescence imaging of single, tetraspanin EVs captured directly from plasma with ExoView®. Consistent with WB, we observed significantly increased plasma CD81+ EV count and cargo at 1d post-injury. The majority of these tetraspanin EVs were smaller than 50 nm based on interferometry and were insufficiently resolved by flow cytometry-based detection. At the injury site, there was enhanced expression of EV biogenesis proteins that were also detected in EVs directly isolated from spinal cord tissue by WB. Surface expression of tetraspanins CD9 and CD63 increased in multiple cell types at the injury site; however, astrocyte CD81 expression uniquely decreased, as demonstrated by flow cytometry. UC-isolated plasma EV microRNA cargo was also significantly altered at 1d post-injury with changes similar to that reported in EVs released by astrocytes after inflammatory stimulation. When injected into the lateral ventricle, plasma EVs from SCI mice increased both pro- and anti-inflammatory gene as well as reactive astrocyte gene expression in the brain cortex. These studies provide the first detailed characterization of plasma EV dynamics after SCI and suggest that plasma EVs may be involved in posttraumatic brain inflammation.
Assuntos
Exossomos , Vesículas Extracelulares , MicroRNAs , Nanopartículas , Traumatismos da Medula Espinal , Animais , CamundongosRESUMO
OBJECTIVES: Respiratory infections in the postacute phase of traumatic brain injury impede optimal recovery and contribute substantially to overall morbidity and mortality. This study investigated bidirectional innate immune responses between the injured brain and lung, using a controlled cortical impact model followed by secondary Streptococcus pneumoniae infection in mice. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: Adult male C57BL/6J mice. INTERVENTIONS: C57BL/6J mice were subjected to sham surgery or moderate-level controlled cortical impact and infected intranasally with S. pneumoniae (1,500 colony-forming units) or vehicle (phosphate-buffered saline) at 3 or 60 days post-injury. MAIN RESULTS: At 3 days post-injury, S. pneumoniae-infected traumatic brain injury mice (TBI + Sp) had a 25% mortality rate, in contrast to no mortality in S. pneumoniae-infected sham (Sham + Sp) animals. TBI + Sp mice infected 60 days post-injury had a 60% mortality compared with 5% mortality in Sham + Sp mice. In both studies, TBI + Sp mice had poorer motor function recovery compared with TBI + PBS mice. There was increased expression of pro-inflammatory markers in cortex of TBI + Sp compared with TBI + PBS mice after both early and late infection, indicating enhanced post-traumatic neuroinflammation. In addition, monocytes from lungs of TBI + Sp mice were immunosuppressed acutely after traumatic brain injury and could not produce interleukin-1ß, tumor necrosis factor-α, or reactive oxygen species. In contrast, after delayed infection monocytes from TBI + Sp mice had higher levels of interleukin-1ß, tumor necrosis factor-α, and reactive oxygen species when compared with Sham + Sp mice. Increased bacterial burden and pathology was also found in lungs of TBI + Sp mice. CONCLUSIONS: Traumatic brain injury causes monocyte functional impairments that may affect the host's susceptibility to respiratory infections. Chronically injured mice had greater mortality following S. pneumoniae infection, which suggests that respiratory infections even late after traumatic brain injury may pose a more serious threat than is currently appreciated.
Assuntos
Lesões Encefálicas Traumáticas/epidemiologia , Monócitos/metabolismo , Infecções Respiratórias/epidemiologia , Infecções Estafilocócicas/epidemiologia , Animais , Lesões Encefálicas Traumáticas/fisiopatologia , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia Estafilocócica , Infecções Respiratórias/mortalidade , Infecções Estafilocócicas/mortalidadeRESUMO
SIGNIFICANCE: Lighting assessments are an important component of low-vision rehabilitation but are rarely studied systematically. Our study indicates that preferred lighting levels support improvements in reading ease and enjoyment, independently of the evaluation technique. To determine preferred illumination level and color temperature, the LuxIQ is quicker to administer and covers broad options of settings. PURPOSE: The purpose of this study was to determine if preferred lighting, as assessed by the LuxIQ versus a standard lighting assessment, leads to better reading outcomes in individuals with low vision. METHODS: Preferred lighting was assessed at home with visually impaired persons (mean age, 75.3 years), using the LuxIQ (n = 18) or a standard technique based on trying out light bulbs of various intensity and color temperature (n = 16). Maximum reading speed and reading acuity were measured before the lighting intervention and then under the preferred lighting conditions. A 1-month telephone follow-up evaluated the (1) compliance with the lighting recommendations and, for those who modified their lighting, (2) their level of satisfaction with the length of reading time and eye strain felt during reading. RESULTS: Compared with usual lighting conditions, most participants preferred a cooler temperature at a higher illuminance level. Neither lighting assessment type appeared to lead to substantially improved objectively measurable reading outcomes. At the 1-month follow-up, 56% of the participants had modified their lighting, having a significant effect on satisfaction (P < .01), independent of assessment method. Of 18 respondents, 16 (87.5%) mentioned that reading was more enjoyable or easier with the lighting modifications. CONCLUSIONS: Both lighting assessment methods lead to comparable results, but the LuxIQ is easier and faster to use. More research is needed to determine whether the LuxIQ is suitable to be incorporated into clinical practice.
Assuntos
Iluminação/métodos , Leitura , Baixa Visão/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Acuidade Visual/fisiologia , Pessoas com Deficiência Visual/reabilitação , Adulto JovemRESUMO
Neuroinflammation is one of the key secondary injury mechanisms triggered by traumatic brain injury (TBI). Microglial activation, a hallmark of brain neuroinflammation, plays a critical role in regulating immune responses after TBI and contributes to progressive neurodegeneration and neurologic deficits following brain trauma. Here we evaluated the role of neutral sphingomyelinase (nSMase) in microglial activation by examining the effects of the nSMase inhibitors altenusin and GW4869 in vitro (using BV2 microglia cells and primary microglia), as well as in a controlled cortical injury (CCI) model in adult male C57BL/6 mice. Pretreatment of altenusin or GW4869 prior to lipopolysaccharide (LPS) stimulation for 4 or 24 hours, significantly downregulated gene expression of the pro-inflammatory mediators TNF-α, IL-1ß, IL-6, iNOS, and CCL2 in microglia and reduced the release of nitric oxide and TNF-α These nSMase inhibitors also attenuated the release of microparticles and phosphorylation of p38 MAPK and ERK1/2. In addition, altenusin pretreatment also reduced the gene expression of multiple inflammatory markers associated with microglial activation after experimental TBI, including TNF-α, IL-1ß, IL-6, iNOS, CCL2, CD68, NOX2, and p22phox Overall, our data demonstrate that nSMase inhibitors attenuate multiple inflammatory pathways associated with microglial activation in vitro and after experimental TBI. Thus, nSMase inhibitors may represent promising therapeutics agents targeting neuroinflammation.
Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , Microglia/metabolismo , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/metabolismo , Animais , Lesões Encefálicas Traumáticas/induzido quimicamente , Lesões Encefálicas Traumáticas/prevenção & controle , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Mediadores da Inflamação/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Depression is a well-recognised effect of long-term treatment with interferon-alpha (IFN-α), a widely used treatment for chronic viral hepatitis and malignancy. In addition to the emotional disturbances, high incidences of painful symptoms such as headache and joint pain have also been reported following IFN-α treatment. The endocannabinoid system plays an important role in emotional and nociceptive processing, however it is unknown whether repeated IFN-α administration induces alterations in this system. The present study investigated nociceptive responding in the IFN-α-induced mouse model of depression and associated changes in the endocannabinoid system. Furthermore, the effects of modulating peripheral endocannabinoid tone on inflammatory pain-related behaviour in the IFN-α model was examined. Repeated IFN-α administration (8000â¯IU/g/day) to male C57/Bl6 mice increased immobility in the forced swim test and reduced sucrose preference, without altering body weight gain or locomotor activity, confirming development of the depressive-like phenotype. There was no effect of repeated IFN-α administration on latency to respond in the hot plate test on day 4 or 7 of treatment, however, formalin-evoked nociceptive behaviour was significantly increased in IFN-α treated mice following 8â¯days of IFN-α administration. 2-Arachidonoyl glycerol (2-AG) levels in the periaqueductal grey (PAG) and rostroventromedial medulla (RVM), and anandamide (AEA) levels in the RVM, were significantly increased in IFN-α-, but not saline-, treated mice following formalin administration. There was no change in endocannabinoid levels in the prefrontal cortex, spinal cord or paw tissue between saline- or IFNα-treated mice in the presence or absence of formalin. Furthermore, repeated IFN-α and/or formalin administration did not alter mRNA expression of genes encoding the endocannabinoid catabolic enzymes (fatty acid amide hydrolase or monoacylglycerol lipase) or endocannabinoid receptor targets (CB1, CB2 or PPARs) in the brain, spinal cord or paw tissue. Intra plantar administration of PF3845 (1⯵g/10⯵l) or MJN110 (1⯵g/10⯵l), inhibitors of AEA and 2-AG catabolism respectively, attenuated formalin-evoked hyperalgesia in IFN-α, but not saline-, treated mice. In summary, increasing peripheral endocannabinoid tone attenuates inflammatory hyperalgesia induced following repeated IFN-α administration. These data provide support for the endocannabinoid system in mediating and modulating heightened pain responding associated with IFNα-induced depression.
Assuntos
Depressão/metabolismo , Endocanabinoides/metabolismo , Interferon-alfa/metabolismo , Amidoidrolases/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Glicerídeos/metabolismo , Hiperalgesia/imunologia , Hiperalgesia/metabolismo , Interferon-alfa/farmacologia , Masculino , Camundongos , Monoacilglicerol Lipases/metabolismo , Nociceptores/efeitos dos fármacos , Nociceptores/metabolismo , Dor/metabolismo , Dor/fisiopatologia , Alcamidas Poli-Insaturadas/metabolismoRESUMO
Toll-like receptor (TLR)3 is a key component of the innate immune response to viral infection. The present study firstly examined whether sex differences exist in TLR3-induced inflammatory, endocrine, and sickness responses. The data revealed that TLR3-induced expression of interferon- or NFkB-inducible genes (IFN-α/ß, IP-10, or TNF-α), either peripherally (spleen) or centrally (hypothalamus), did not differ between male and female rats, with the exception of TLR3-induced IFN-α expression in the spleen of female, but not male, rats 8 hr post TLR3 activation. Furthermore, TLR3 activation increased plasma corticosterone levels, induced fever, and reduced locomotor activity and body weight - effects independent of sex. Thus, the acute-phase inflammatory, endocrine, and sickness responses to TLR3 activation exhibit minimal sex-related differences. A further aim of this study was to examine whether enhancing endocannabinoid tone - namely, 2-arachidonylglycerol (2-AG) or N-arachidonoylethanolamine (AEA), exhibited similar effects on TLR3-induced inflammatory responses in male versus female rats. Systemic administration of the monoacylglycerol lipase (MAGL) inhibitor MJN110 and subsequent increases in 2-AG levels did not alter the TLR3-induced increase in IP-10, IRF7, or TNF-α expression in the spleen or the hypothalamus of male or female rats. In contrast, the fatty acid amide hydrolase (FAAH) inhibitor URB597 increased levels of AEA and related N-acylethanolamines, an effect associated with the attenuation of TLR3-induced inflammatory responses in the hypothalamus, but not the spleen, of male and female rats. These data support a role for FAAH, but not MAGL, substrates in the modulation of TLR3-induced neuroinflammatory responses, effects independent of sex.
Assuntos
Amidoidrolases/antagonistas & inibidores , Monoacilglicerol Lipases/antagonistas & inibidores , Receptor 3 Toll-Like/imunologia , Amidas , Amidoidrolases/imunologia , Amidoidrolases/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Temperatura Corporal/efeitos dos fármacos , Carbamatos/farmacologia , Quimiocina CXCL10/metabolismo , Corticosterona/sangue , Endocanabinoides/metabolismo , Estradiol/metabolismo , Etanolaminas/metabolismo , Feminino , Glicerídeos/metabolismo , Fatores Imunológicos/imunologia , Interferons/metabolismo , Masculino , Monoacilglicerol Lipases/imunologia , NF-kappa B/metabolismo , Ácidos Oleicos/metabolismo , Ácidos Palmíticos/metabolismo , Poli I-C/farmacologia , Alcamidas Poli-Insaturadas , Ratos , Ratos Sprague-Dawley , Fatores Sexuais , Transdução de Sinais/imunologia , Succinimidas/farmacologia , Receptor 3 Toll-Like/metabolismoRESUMO
Alkene monooxygenases (MOs) are soluble di-iron-containing enzymes found in bacteria that grow on alkenes. Here, we report improved heterologous expression systems for the propene MO (PmoABCD) and ethene MO (EtnABCD) from Mycobacterium chubuense strain NBB4. Strong functional expression of PmoABCD and EtnABCD was achieved in Mycobacterium smegmatis mc2155, yielding epoxidation activities (62 and 27 nmol/min/mg protein, respectively) higher than any reported to date for heterologous expression of a di-iron MO system. Both PmoABCD and EtnABCD were specialized for the oxidation of gaseous alkenes (C2 to C4), and their activity was much lower on liquid alkenes (C5 to C8). Despite intensive efforts to express the complete EtnABCD enzyme in Escherichia coli, this was not achieved, although recombinant EtnB and EtnD proteins could be purified individually in soluble form. The biochemical function of EtnD as an oxidoreductase was confirmed (1.36 µmol cytochrome c reduced/min/mg protein). Cloning the EtnABCD gene cluster into Pseudomonas putida KT2440 yielded detectable epoxidation of ethene (0.5 nmol/min/mg protein), and this could be stimulated (up to 1.1 nmol/min/mg protein) by the coexpression of cpn60 chaperonins from either Mycobacterium spp. or E. coli Successful expression of the ethene MO in a Gram-negative host was validated by both whole-cell activity assays and peptide mass spectrometry of induced proteins seen on SDS-PAGE gels.IMPORTANCE Alkene MOs are of interest for their potential roles in industrial biocatalysis, most notably for the stereoselective synthesis of epoxides. Wild-type bacteria that grow on alkenes have high activities for alkene oxidation but are problematic for biocatalysis, since they tend to consume the epoxide products. Using recombinant biocatalysts is the obvious alternative, but a major bottleneck is the low activities of recombinant alkene MOs. Here, we provide new high-activity recombinant biocatalysts for alkene oxidation, and we provide insights into how to further improve these systems.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Expressão Gênica , Mycobacterium smegmatis/genética , Mycobacterium/enzimologia , Oxigenases/genética , Pseudomonas putida/genética , Alcenos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citocromos c , Escherichia coli/metabolismo , Etilenos/metabolismo , Cinética , Mycobacterium/genética , Mycobacterium smegmatis/metabolismo , Oxigenases/química , Oxigenases/metabolismo , Pseudomonas putida/metabolismoRESUMO
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
Aberrant activation of toll-like receptors (TLRs), key components of the innate immune system, has been proposed to underlie and exacerbate a range of central nervous system disorders. Increasing evidence supports a role for the endocannabinoid system in modulating inflammatory responses including those mediated by TLRs, and thus this system may provide an important treatment target for neuroinflammatory disorders. However, the effect of modulating endocannabinoid tone on TLR-induced neuroinflammation in vivo and associated behavioural changes is largely unknown. The present study examined the effect of inhibiting fatty acid amide hydrolyase (FAAH), the primary enzyme responsible for the metabolism of anandamide (AEA), in vivo on TLR4-induced neuroimmune and behavioural responses, and evaluated sites and mechanisms of action. Systemic administration of the FAAH inhibitor PF3845 increased levels of AEA, and related FAAH substrates N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA), in the frontal cortex and hippocampus of rats, an effect associated with an attenuation in the expression of pro- and anti-inflammatory cytokines and mediators measured 2hrs following systemic administration of the TLR4 agonist, lipopolysaccharide (LPS). These effects were mimicked by central i.c.v. administration of PF3845, but not systemic administration of the peripherally-restricted FAAH inhibitor URB937. Central antagonism of TRPV1 significantly attenuated the PF3845-induced decrease in IL-6 expression, effects not observed following antagonism of CB1, CB2, PPARα, PPARγ or GPR55. LPS-induced a robust sickness-like behavioural response and increased the expression of markers of glial activity and pro-inflammatory cytokines over 24hrs. Systemic administration of PF3845 modulated the TLR4-induced expression of neuroimmune mediators and anhedonia without altering acute sickness behaviour. Overall, these findings support an important role for FAAH substrates directly within the brain in the regulation of TLR4-associated neuroinflammation and highlight a role for TRPV1 in partially mediating these effects.
Assuntos
Amidoidrolases/antagonistas & inibidores , Encefalite/tratamento farmacológico , Comportamento de Doença/efeitos dos fármacos , Piperidinas/uso terapêutico , Piridinas/uso terapêutico , Canais de Cátion TRPV/metabolismo , Receptor 4 Toll-Like/agonistas , Animais , Encefalite/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Piperidinas/administração & dosagem , Piridinas/administração & dosagem , Ratos , Ratos Sprague-DawleyRESUMO
Prehospital dosing errors affect approximately 56,000 US children yearly. To decrease these errors, barriers, enablers and solutions from the paramedic (EMT-P) and medical director (MD) standpoint need to be understood. We conducted a mixed-methods study of EMT-P and MDs in Michigan utilizing focus groups (FG). FGs were held at EMS agencies and state EMS conferences. Questions focused on the drug dose delivery process, barriers and enablers to correct dosing and possible solutions to decrease errors. Responses were coded by the research team for themes and number of response mentions. Participants completed a pre-FG survey on pediatric experience and agency characteristics. There were 35 EMT-P and 9 MD participants: 43% of EMT-Ps had been practicing > 10 years, 11% had been practicing < 1 year; and 25% reported they had not administered a drug dose to a child in the last 12 months. EMT-Ps who were "very comfortable" with their ability to administer a correct drug dose to infants, toddlers, school-aged, and adolescents were: 5%, 7%, 10%, and 54%, respectively. FGs identified themes of: difficulty obtaining weight, infrequent pediatric encounters, infrequent/inadequate pediatric training, difficulties with drug packaging, drug bags that were not "EMS friendly," difficulty with drug calculations, and lack of dosing aids. Simplification of dose delivery, an improved length based tape for EMS, pediatric checklists, and dose cards in mL were given as solutions. This mixed-methods study identified barriers and potential solutions to reducing prehospital pediatric drug dosing errors. Solutions should be thoroughly tested prior to implementation.
Assuntos
Pessoal Técnico de Saúde , Serviços Médicos de Emergência/normas , Erros de Medicação/prevenção & controle , Erros de Medicação/estatística & dados numéricos , Adolescente , Criança , Pré-Escolar , Feminino , Grupos Focais , Humanos , Lactente , Recém-Nascido , Masculino , Michigan , Fatores de RiscoRESUMO
Veterinary medical school challenges students academically and personally, and some students report depression and anxiety at rates higher than the general population and other medical students. This study describes changes in veterinary medical student self-esteem (SE) over four years of professional education, attending to differences between high and low SE students and the characteristics specific to low SE veterinary medical students. The study population was students enrolled at the Michigan State University College of Veterinary Medicine from 2006 to 2012. We used data from the annual anonymous survey administered college-wide that is used to monitor the curriculum and learning environment. The survey asked respondents to rate their knowledge and skill development, learning environment, perceptions of stress, skill development, and SE. Participants also provided information on their academic performance and demographics. A contrasting groups design was used: high and low SE students were compared using logistic regression to identify factors associated with low SE. A total of 1,653 respondents met inclusion criteria: 789 low SE and 864 high SE students. The proportion of high and low SE students varied over time, with the greatest proportion of low SE students during the second-year of the program. Perceived stress was associated with low SE, whereas perceived supportive learning environment and skill development were associated with high SE. These data have provided impetus for curricular and learning environment changes to enhance student support. They also provide guidance for additional research to better understand various student academic trajectories and their implications for success.
Assuntos
Adaptação Psicológica , Transtorno Depressivo/psicologia , Educação em Veterinária , Estudantes de Medicina/psicologia , Adulto , Currículo , Transtorno Depressivo/prevenção & controle , Feminino , Humanos , Masculino , Michigan , Projetos Piloto , Psicometria , Autoimagem , Adulto JovemRESUMO
BACKGROUND: We describe the results of cognitive interviews to refine the "Making Choices©" Decision Aid (DA) for shared decision-making (SDM) about stress testing in patients with stable coronary artery disease (CAD). METHODS: We conducted a systematic development process to design a DA consistent with International Patient Decision Aid Standards (IPDAS) focused on Alpha testing criteria. Cognitive interviews were conducted with ten stable CAD patients using the "think aloud" interview technique to assess the clarity, usefulness, and design of each page of the DA. RESULTS: Participants identified three main messages: 1) patients have multiple options based on stress tests and they should be discussed with a physician, 2) take care of yourself, 3) the stress test is the gold standard for determining the severity of your heart disease. Revisions corrected the inaccurate assumption of item number three. CONCLUSIONS: Cognitive interviews proved critical for engaging patients in the development process and highlighted the necessity of clear message development and use of design principles that make decision materials easy to read and easy to use. Cognitive interviews appear to contribute critical information from the patient perspective to the overall systematic development process for designing decision aids.
Assuntos
Doença da Artéria Coronariana/diagnóstico , Tomada de Decisões , Técnicas de Apoio para a Decisão , Teste de Esforço/normas , Folhetos , Idoso , Feminino , Humanos , Entrevista Psicológica/métodos , Masculino , Pessoa de Meia-Idade , Participação do Paciente/métodosRESUMO
A sizable proportion of patients with mild traumatic brain injury (mTBI) have persistent symptoms and functional impairments months to years following injury. This phenomenon is continually observed despite an explosion of research and interest in improving mTBI clinical outcomes over the last two decades. All pharmacological clinical trials to date have failed to demonstrate improved outcomes for mTBI. One possible explanation for these continued failures is an overly myopic approach to treating mTBI (i.e., testing the effect of a single drug with a specific mechanism on a group of people with highly heterogenous injuries). Clinical presentation and prognosis of mTBI vary considerably between patients, and yet we continue to assess group-level effects of a homogenized treatment. We need to utilize an equally complex treatment approach to match the extraordinary complexity of the human brain. Dynamical systems theory has been used to describe systems composed of multiple subsystems who function somewhat independently but are ultimately interconnected. This theory was popularized in the motor control literature as an overarching framework for how the mind and body connect to interact and move through the environment. However, the human body can be viewed as a dynamical system composed of multiple subsystems (i.e., organ systems) who have isolated functions, which are also codependent on the health and performance of other interconnected organ systems. In this perspective piece, we will use the example of mTBI in the obese patient to demonstrate how broadening our approach to treatment of the individual (and not necessarily the injury) may ultimately yield improved outcomes. Furthermore, we will explore clinical and pre-clinical evidence demonstrating multiple system interactions in the context of obesity and TBI and discuss how expanding our understanding of the mechanistic interplay between multiple organ systems may ultimately provide a more personalized treatment approach for this mTBI patient subpopulation.
RESUMO
Activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a moderating factor between obesity and cognitive impairment in animals, but this has never been tested in humans following mild traumatic brain injury (mTBI). This is a retrospective cohort analysis of subjects enrolled at a single level 1 trauma center (n = 172). Participants completed Trail Making Test Part A and B (TMT-A and B) at six- and twelve-months, Blood samples were obtained within 24 h of mTBI and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, interleukin-18 (IL-18), and IL-1ß were assayed. Obese participants (BMI = 30-34.9) were associated with higher IL-18 (p = 0.03) and IL-1ß (p = 0.05) and severely obese participants (BMI > 35.0) were associated with higher IL-1ß (p = 0.005) than healthy weight participants. IL-1ß was associated with TMT-A at six- (p = 0.01) and twelve-months (p = 0.03) and TMT-B at twelve-months (p = 0.046). The interaction of severely obese BMI and IL-1ß was associated with TMT-B at six- (p = 0.049) and twelve-months (p = 0.02). ASC (p = 0.03) and the interaction of ASC with severely obese BMI was associated with TMTB at six- (p = 0.02) and twelve-months (p = 0.02). Obesity may augment acute inflammasome response to mTBI and influence worse long-term cognitive outcomes up to one-year post-injury.