RESUMEN
The molecular basis for circadian dependency in stroke due to subarachnoid hemorrhagic stroke (SAH) remains unclear. We reasoned that microglial erythrophagocytosis, crucial for SAH response, follows a circadian pattern involving carbon monoxide (CO) and CD36 surface expression. The microglial BV-2 cell line and primary microglia (PMG) under a clocked medium change were exposed to blood ± CO (250 ppm, 1 h) in vitro. Circadian dependency and the involvement of CD36 were analyzed in PMG isolated from control mice and CD36-/- mice and by RNA interference targeting Per-2. In vivo investigations, including phagocytosis, vasospasm, microglia activation and spatial memory, were conducted in an SAH model using control and CD36-/- mice at different zeitgeber times (ZT). In vitro, the surface expression of CD36 and its dependency on CO and phagocytosis occurred with changed circadian gene expression. CD36-/- PMG exhibited altered circadian gene expression, phagocytosis and impaired responsiveness to CO. In vivo, control mice with SAH demonstrated circadian dependency in microglia activation, erythrophagocytosis and CO-mediated protection at ZT2, in contrast to CD36-/- mice. Our study indicates that circadian rhythmicity modulates microglial activation and subsequent CD36-dependent phagocytosis. CO altered circadian-dependent neuroprotection and CD36 induction, determining the functional outcome in a hemorrhagic stroke model. This study emphasizes how circadian rhythmicity influences neuronal damage after neurovascular events.
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Accidente Cerebrovascular Hemorrágico , Linfohistiocitosis Hemofagocítica , Hemorragia Subaracnoidea , Ratones , Animales , Microglía/metabolismo , Monóxido de Carbono/metabolismo , Neuroprotección , Fagocitosis/fisiología , Hemorragia Subaracnoidea/metabolismoRESUMEN
BACKGROUND: As the COVID-19 pandemic strains healthcare systems worldwide, finding predictive markers of severe courses remains urgent. Most research so far was limited to selective questions hindering general assumptions for short- and long-term outcome. METHODS: In this prospective single-center biomarker study, 47 blood- and 21 bronchoalveolar lavage (BAL) samples were collected from 47 COVID-19 intensive care unit (ICU) patients upon admission. Expression of inflammatory markers toll-like receptor 3 (TLR3), heme oxygenase-1 (HO-1), interleukin (IL)-6, IL-8, leukocyte counts, procalcitonin (PCT) and carboxyhemoglobin (CO-Hb) was compared to clinical course. Clinical assessment comprised acute local organ damage, acute systemic damage, mortality and outcome after 6 months. RESULTS: PCT correlated with acute systemic damage and was the best predictor for quality of life (QoL) after 6 months (r = - 0.4647, p = 0.0338). Systemic TLR3 negatively correlated with impaired lung function (ECMO/ECLS: r = - 0.3810, p = 0.0107) and neurological short- (RASS mean: r = 0.4474, p = 0.0023) and long-term outcome (mRS after 6 m: r = - 0.3184, p = 0.0352). Systemic IL-8 correlated with impaired lung function (ECMO/ECLS: r = 0.3784, p = 0.0161) and neurological involvement (RASS mean: r = - 0.5132, p = 0.0007). IL-6 in BAL correlated better to the clinical course than systemic IL-6. Using three multivariate regression models, we describe prediction models for local and systemic damage as well as QoL. CO-Hb mean and max were associated with higher mortality. CONCLUSIONS: Our predictive models using the combination of Charlson Comorbidity Index, sex, procalcitonin, systemic TLR3 expression and IL-6 and IL-8 in BAL were able to describe a broad range of clinically relevant outcomes in patients with severe COVID-19-associated ARDS. Using these models might proof useful in risk stratification and predicting disease course in the future. Trial registration The trial was registered with the German Clinical Trials Register (Trial-ID DRKS00021522, registered 22/04/2020).
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COVID-19 , Síndrome de Dificultad Respiratoria , Humanos , COVID-19/complicaciones , Calidad de Vida , Receptor Toll-Like 3 , Interleucina-6 , Interleucina-8 , Polipéptido alfa Relacionado con Calcitonina , Estudios Prospectivos , Pandemias , Inflamación , Síndrome de Dificultad Respiratoria/etiología , Progresión de la EnfermedadRESUMEN
BACKGROUND: Neuraxial access is necessary for an array of procedures in anaesthesia, interventional pain medicine and neurosurgery. The commonly used anatomical landmark technique is challenging and requires practical experience. OBJECTIVE: We aimed to evaluate the technical feasibility of an augmented reality-guided approach for neuraxial access and tested the hypothesis that its use would improve success as the primary outcome. As secondary outcomes, we measured accuracy and the procedural duration compared with the classical landmark approach. DESIGN: A randomised phantom-based study. SETTING: The three-dimensional image of a thoracolumbar phantom spine model with the surrounding soft tissue was created with a neurosurgical planning workstation and ideal trajectories to the epidural space on the levels T10-L1 were planned using a paramedian approach. Both the three-dimensional holographic image of the spine and the trajectories were transferred to an augmented reality-headset. Four probands (two anaesthesiologists, one neuroradiologist and one stereotactic neurosurgeon) performed 20 attempts, 10 each of either conventional landmark or augmented reality-guided epidural punctures, where anatomical level, side and sequence of modality were all randomised. OUTCOME MEASURES: Accuracy was assessed by measuring Euclidean distance and lateral deviation from the predefined target point. Success of epidural puncture on the first attempt was compared between the conventional and the augmented reality-guided approaches. RESULTS: Success was achieved in 82.5% of the attempts using augmented reality technique, compared with 40% with the conventional approach [ P â=â0.0002, odds ratio (OR) for success: 7.07]. Euclidean distance (6.1 vs. 12âmm, P â<â0.0001) and lateral deviation (3.7 vs. 9.2âmm, P â<â0.0001) were significantly smaller using augmented reality. Augmented reality-guided puncture was significantly faster than with the conventional landmark approach (52.5 vs. 67.5âs, P â=â0.0015). CONCLUSION: Augmented reality guidance significantly improved the accuracy and success in an experimental phantom model of epidural puncture. With further technical development, augmented reality guidance might prove helpful in anatomically challenging neuraxial procedures.
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Realidad Aumentada , Humanos , Espacio Epidural/diagnóstico por imagen , Fantasmas de Imagen , Punciones/métodosRESUMEN
PURPOSE: Six to 19% of critically ill COVID-19 patients display circulating auto-antibodies against type I interferons (IFN-AABs). Here, we establish a clinically applicable strategy for early identification of IFN-AAB-positive patients for potential subsequent clinical interventions. METHODS: We analyzed sera of 430 COVID-19 patients from four hospitals for presence of IFN-AABs by ELISA. Binding specificity and neutralizing activity were evaluated via competition assay and virus-infection-based neutralization assay. We defined clinical parameters associated with IFN-AAB positivity. In a subgroup of critically ill patients, we analyzed effects of therapeutic plasma exchange (TPE) on the levels of IFN-AABs, SARS-CoV-2 antibodies and clinical outcome. RESULTS: The prevalence of neutralizing AABs to IFN-α and IFN-ω in COVID-19 patients from all cohorts was 4.2% (18/430), while being undetectable in an uninfected control cohort. Neutralizing IFN-AABs were detectable exclusively in critically affected (max. WHO score 6-8), predominantly male (83%) patients (7.6%, 18/237 for IFN-α-AABs and 4.6%, 11/237 for IFN-ω-AABs in 237 patients with critical COVID-19). IFN-AABs were present early post-symptom onset and at the peak of disease. Fever and oxygen requirement at hospital admission co-presented with neutralizing IFN-AAB positivity. IFN-AABs were associated with lower probability of survival (7.7% versus 80.9% in patients without IFN-AABs). TPE reduced levels of IFN-AABs in three of five patients and may increase survival of IFN-AAB-positive patients compared to those not undergoing TPE. CONCLUSION: IFN-AABs may serve as early biomarker for the development of severe COVID-19. We propose to implement routine screening of hospitalized COVID-19 patients for rapid identification of patients with IFN-AABs who most likely benefit from specific therapies.
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COVID-19 , Interferón Tipo I , Anticuerpos Neutralizantes , Autoanticuerpos , COVID-19/diagnóstico , Enfermedad Crítica , Femenino , Humanos , Interferón-alfa/uso terapéutico , Masculino , Oxígeno , SARS-CoV-2RESUMEN
BACKGROUND: Red blood cell-induced cerebral inflammation and toxicity has been shown to be attenuated by induction of the heme-catalyzing enzyme, hemoxygenase-1 (HO-1), in animal models of subarachnoid hemorrhage (SAH). Although inflammatory mechanisms leading to secondary neuronal injury in SAH are becoming increasingly well understood, markers of cerebral inflammation have so far not been implemented in clinical prediction models of SAH. METHODS: In this biomarker observational study, HO-1 messenger ribonucleic acid (mRNA) expression levels were determined in cerebrospinal fluid (CSF) and blood of 66 patients with aneurysmal SAH on days 1, 7, and 14 after the SAH event. HO-1 mRNA expression was determined via real time polymerase chain reaction (PCR), and relative expression changes were quantified in comparison with expression levels in nonhemorrhagic control CSF. Subarachnoid blood burden, as well as presence of vasospasm and delayed cerebral ischemia (DCI), were recorded. Short and long-term clinical outcomes were assessed using the Modified Rankin Scale at discharge and 1 year after the SAH event. RESULTS: CSF HO-1 expression levels showed a significant increase over the 14-day observation period (p < 0.001, F = 22.53) and correlated with intracranial hematoma burden (ρ = 0.349, p = 0.025). In multivariate analyses, CSF HO-1 expression levels did not reach significance as independent predictors of outcome. Vasospasm on computed tomographic angiography was associated with lower CSF HO-1 expression levels on day 7 after SAH (n = 53, p = 0.010), whereas patients with DCI showed higher CSF HO-1 expression levels on day 14 after SAH (n = 21, p = 0.009). CONCLUSIONS: HO-1 expression in CSF in patients with SAH follows a distinct temporal induction pattern and is dependent on intracranial hematoma burden. CSF HO-1 expression was unable to predict functional outcome. Associations of early low HO-1 expression with vasospasm and late elevated HO-1 expression with DCI may point to detrimental effects of late HO-1 induction, warranting the need for further investigation in a larger study population.
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Isquemia Encefálica , Hemorragia Subaracnoidea , Vasoespasmo Intracraneal , Isquemia Encefálica/complicaciones , Infarto Cerebral/complicaciones , Humanos , Hemorragia Subaracnoidea/complicaciones , Hemorragia Subaracnoidea/metabolismo , Tomografía Computarizada por Rayos X , Vasoespasmo Intracraneal/complicacionesRESUMEN
Microglial erythrophagocytosis is crucial in injury response to hemorrhagic stroke. We hypothesized that regulation of microglial erythrophagocytosis via HO-1/CO depends on a pathway involving reactive oxygen species (ROS) and CD36 surface-expression. The microglial BV-2 cell line and primary microglia (PMG) were incubated +/-blood and +/-CO-exposure. PMG isolated from tissue-specific HO-1-deficient (LyzM-Cre-Hmox1 fl/fl ) and CD36 -/- mice or siRNA against AMPK (AMP-activated protein kinase) were used to test our hypothesis. In a murine subarachnoid hemorrhage (SAH) model, we compared neuronal injury in wild-type and CD36 -/- mice. Readouts included vasospasm, microglia activation, neuronal apoptosis, and spatial memory. We observed increased microglial HO-1-expression after blood-exposure. A burst in ROS-production was seen after CO-exposure, which led to increased amounts of phosphorylated AMPK with subsequently enhanced CD36 surface-expression. Naïve PMG from LyzM-Cre-Hmox1 fl/fl mice showed reduced ROS-production and CD36 surface-expression and failed to respond to CO with increased CD36 surface-expression. Lack of HO-1 and CD36 resulted in reduced erythrophagocytosis that could not be rescued with CO. Erythrophagocytosis was enhanced in BV-2 cells in the presence of exogenous CO, which was abolished in cells treated with siRNA to AMPK. CD36 -/- mice subjected to SAH showed enhanced neuronal cell death, which resulted in impaired spatial memory function. We demonstrate that microglial phagocytic function partly depends on a pathway involving HO-1 with changes in ROS-production, phosphorylated AMPK, and surface expression of CD36. CD36 was identified as a crucial component in blood clearance after hemorrhage that ultimately determines neuronal outcome. These results demand further investigations studying the potential neuroprotective properties of CO.
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Microglía , Hemorragia Subaracnoidea , Proteínas Quinasas Activadas por AMP , Animales , Monóxido de Carbono , Hemo-Oxigenasa 1 , Ratones , ARN Interferente Pequeño , Especies Reactivas de OxígenoRESUMEN
OBJECTIVES: Neurologic damage following cardiac arrest remains a major burden for modern resuscitation medicine. Cardiopulmonary resuscitation with extracorporeal circulatory support holds the potential to reduce morbidity and mortality. Furthermore, the endogenous gasotransmitter carbon monoxide attracts attention in reducing cerebral injury. We hypothesize that extracorporeal resuscitation with additional carbon monoxide application reduces neurologic damage. DESIGN: Randomized, controlled animal study. SETTING: University research laboratory. SUBJECTS: Landrace-hybrid pigs. INTERVENTIONS: In a porcine model, carbon monoxide was added using a novel extracorporeal releasing system after resuscitation from cardiac arrest. MEASUREMENTS AND MAIN RESULTS: As markers of cerebral function, neuromonitoring modalities (somatosensory-evoked potentials, cerebral oximetry, and transcranial Doppler ultrasound) were used. Histopathologic damage and molecular markers (caspase-3 activity and heme oxygenase-1 expression) were analyzed. Cerebral oximetry showed fast rise in regional oxygen saturation after carbon monoxide treatment at 0.5 hours compared with extracorporeal resuscitation alone (regional cerebral oxygen saturation, 73% ± 3% vs 52% ± 8%; p < 0.05). Median nerve somatosensory-evoked potentials showed improved activity upon carbon monoxide treatment, whereas post-cardiac arrest cerebral perfusion differences were diminished. Histopathologic damage scores were reduced compared with customary resuscitation strategies (hippocampus: sham, 0.4 ± 0.2; cardiopulmonary resuscitation, 1.7 ± 0.4; extracorporeal cardiopulmonary resuscitation, 2.3 ± 0.2; extracorporeal cardiopulmonary resuscitation with carbon monoxide application [CO-E-CPR], 0.9 ± 0.3; p < 0.05). Furthermore, ionized calcium-binding adaptor molecule 1 staining revealed reduced damage patterns upon carbon monoxide treatment. Caspase-3 activity (cardiopulmonary resuscitation, 426 ± 169 pg/mL; extracorporeal cardiopulmonary resuscitation, 240 ± 61 pg/mL; CO-E-CPR, 89 ± 26 pg/mL; p < 0.05) and heme oxygenase-1 (sham, 1 ± 0.1; cardiopulmonary resuscitation, 2.5 ± 0.4; extracorporeal cardiopulmonary resuscitation, 2.4 ± 0.2; CO-E-CPR, 1.4 ± 0.2; p < 0.05) expression were reduced after carbon monoxide exposure. CONCLUSIONS: Carbon monoxide application during extracorporeal resuscitation reduces injury patterns in neuromonitoring and decreases histopathologic cerebral damage by reducing apoptosis. This may lay the basis for further clinical translation of this highly salutary substance.
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Encéfalo , Monóxido de Carbono , Reanimación Cardiopulmonar , Oxigenación por Membrana Extracorpórea , Paro Cardíaco , Animales , Masculino , Encéfalo/irrigación sanguínea , Monóxido de Carbono/metabolismo , Monóxido de Carbono/uso terapéutico , Reanimación Cardiopulmonar/métodos , Circulación Cerebrovascular/fisiología , Oxigenación por Membrana Extracorpórea/métodos , Paro Cardíaco/terapia , Porcinos , Resultado del TratamientoRESUMEN
BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) is associated with a temporal pattern of stroke incidence. We hypothesized that natural oscillations in gene expression controlling circadian rhythm affect the severity of neuronal injury. We moreover predict that heme oxygenase-1 (HO-1/Hmox1) and its product carbon monoxide (CO) contribute to the restoration of rhythm and neuroprotection. METHODS: Murine SAH model was used where blood was injected at various time points of the circadian cycle. Readouts included circadian clock gene expression, locomotor activity, vasospasm, neuroinflammatory markers, and apoptosis. In addition, cerebrospinal fluid and peripheral blood leukocytes from SAH patients and controls were analyzed for clock gene expression. RESULTS: Significant elevations in the clock genes Per-1, Per-2, and NPAS-2 were observed in the hippocampus, cortex, and suprachiasmatic nucleus in mice subjected to SAH at zeitgeber time (ZT) 12 when compared with ZT2. Clock gene expression amplitude correlated with basal expression of HO-1, which was also significantly greater at ZT12. SAH animals showed a significant reduction in cerebral vasospasm, neuronal apoptosis, and microglial activation at ZT12 compared with ZT2. In animals with myeloid-specific HO-1 deletion (Lyz-Cre-Hmox1fl/fl ), Per-1, Per-2, and NPAS-2 expression was reduced in the suprachiasmatic nucleus, which correlated with increased injury. Treatment with low-dose CO rescued Lyz-Cre-Hmox1fl/fl mice, restored Per-1, Per-2, and NPAS-2 expression, and reduced neuronal apoptosis. CONCLUSIONS: Clock gene expression regulates, in part, the severity of SAH and requires myeloid HO-1 activity to clear the erythrocyte burden and inhibit neuronal apoptosis. Exposure to CO rescues the loss of HO-1 and thus merits further investigation in patients with SAH.
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Monóxido de Carbono/metabolismo , Ritmo Circadiano/genética , Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Proteínas de la Membrana/genética , Hemorragia Subaracnoidea/genética , Factores de Transcripción ARNTL/genética , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas CLOCK/genética , Líquido Cefalorraquídeo/metabolismo , Hemo-Oxigenasa 1/metabolismo , Humanos , Inmunohistoquímica , Inflamación , Leucocitos/metabolismo , Locomoción , Proteínas de la Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas Circadianas Period/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la Enfermedad , Núcleo Supraquiasmático/metabolismo , Vasoespasmo IntracranealRESUMEN
BACKGROUND: The endogenously produced gaseous molecule carbon monoxide is able to promote organ protection after ischemia-reperfusion injuries (IRI). The impact of carbon monoxide releasing molecules (CORM) regarding inflammation in neuronal tissues has not been studied in detail. In this investigation, we aimed to analyze the effects of the CORM ALF-186 on neuro-inflammation and hypothesized that the soluble guanylate cyclase (sGC) is playing a decisive role. METHODS: Retinal ischemia-reperfusion injury was performed for 60 min in Sprague-Dawley rats. Thereafter, the CORM ALF-186 (10 mg/kg) in the presence or absence of the sGC inhibitor ODQ was injected via a tail vein. Retinal tissue was harvested 24 h later to analyze mRNA or protein expression of sGC-ß1 subunit, transcription factors NF-κB and CREB, the inflammatory cytokines TNF-α and IL-6, as well as the heat shock proteins (HSP) HSP-70 and HSP-90. Immunohistochemistry was performed on frozen sections of the retina. The overall neuroprotective effect of ALF-186 was assessed by counting fluorogold-pre-labeled retinal ganglion cells (RGC) 7 days after IRI. RESULTS: Ischemia-reperfusion mediated loss of vital RGC was attenuated by the administration of ALF-186 after injury. ALF-186 treatment after IRI induced sGC-ß1 leading to a decreased NF-κB and CREB phosphorylation. Consecutively, ALF-186 mitigated IRI induced TNF-α and IL-6 expression in the retina and in the rats' serum. Moreover, ALF-186 attenuated heat shock protein 70 (Hsp-70) while increasing Hsp-90. The sGC-inhibitor ODQ attenuated the anti-inflammatory effects of ALF-186 and increased retinal loss of ganglion cells. These results were confirmed by immunohistochemistry. CONCLUSION: The CORM ALF-186 protected RGC from IRI induced loss. Furthermore, ALF-186 reduced IRI mediated neuroinflammation in the retina and in the serum by activating sGC. Inhibition of sGC stopped the beneficial and protective effects of ALF-186. ALF-186 may present a promising therapeutic alternative in treating inflammation after neuronal IRI.
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Antiinflamatorios/uso terapéutico , Complejos de Coordinación/uso terapéutico , Isquemia/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Daño por Reperfusión/tratamiento farmacológico , Guanilil Ciclasa Soluble/metabolismo , Animales , Antiinflamatorios/farmacología , Monóxido de Carbono/metabolismo , Complejos de Coordinación/farmacología , Femenino , Isquemia/metabolismo , Isquemia/patología , Masculino , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patologíaRESUMEN
We report a repositioning maneuver of a dislocated Avalon Elite dual lumen catheter during ongoing veno-venous ECMO support. The inferior tip of the catheter dislocated into a liver vein, which was accompanied by a dramatic decrease in pump flow. After standard repositioning maneuvers under transthoracic echocardiographic guidance had failed, a special guiding sheath was inserted into the main lumen through a Y-connector with a valve. Over this valve, a stiff wire could be placed into the inferior vena cava to help guiding the catheter back into the correct position.
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Catéteres/efectos adversos , Oxigenación por Membrana Extracorpórea/instrumentación , Migración de Cuerpo Extraño/diagnóstico , Venas Hepáticas , Sistemas de Atención de Punto , Síndrome de Dificultad Respiratoria/cirugía , Cánula , Ecocardiografía , Humanos , Persona de Mediana EdadRESUMEN
BACKGROUND: Propofol, midazolam and ketamine are widely used in today's anaesthesia practice. Both neuroprotective and neurotoxic effects have been attributed to all three agents. OBJECTIVE: To establish whether propofol, midazolam and ketamine in the same neuronal injury model exert neuroprotective effects on injured neurones in vitro and in vivo by modulation of the Toll-like receptor 4-nuclear factor kappa-light-chain-enhancer of activated B cells (TLR-4-NF-κB) pathway. DESIGN AND SETTING: Cell-based laboratory (nâ=â6 repetitions per experiment) and animal (nâ=â6 per group) studies using a neuronal cell line (SH-SY5Y cells) and adult Sprague-Dawley rats. INTERVENTIONS: Cells were exposed to oxygen-glucose deprivation before or after treatment using escalating, clinically relevant doses of propofol, midazolam and ketamine. In animals, retinal ischaemia (60âmin) was induced followed by reperfusion and randomised treatment with saline or propofol. MAIN OUTCOME MEASURES: Neuronal cell death was determined using flow-cytometry (mitochondrial membrane potential) and lactate dehydrogenase (LDH) release. Nuclear factor NF-κB and hypoxia-inducible factor 1 α-activity were analysed by DNA-binding ELISA, expression of NF-κB-dependent genes and TLR-4 by luciferase-assay and flow-cytometry, respectively. In animals, retinal ganglion cell density, caspase-3 activation and gene expression (TLR-4, NF-κB) were used to determine in vivo effects of propofol. Results were compared using ANOVA (Analysis of Variance) and t test. A P value less than 0.05 was considered statistically significant. RESULTS: Post-treatment with clinically relevant concentrations of propofol (1 to 10âµgâml) preserved the mitochondrial membrane potential in oxygen-glucose deprivation-injured cells by 54% and reduced LDH release by 21%. Propofol diminished TLR-4 surface expression and preserved the DNA-binding activity of the protective hypoxia-inducible factor 1 α transcription factor. DNA-binding and transcriptional NF-κB-activity were inhibited by propofol. Neuronal protection and inhibition of TLR-4-NF-κB signalling were not consistently seen with midazolam or ketamine. In vivo, propofol treatment preserved rat retinal ganglion cell densities (cellsâmm, saline 1504â±â251 vs propofol 2088â±â144, Pâ=â0.0001), which was accompanied by reduced neuronal caspase-3, TLR-4 and NF-κB expression. CONCLUSION: Propofol, but neither midazolam nor ketamine, provides neuroprotection to injured neuronal cells via inhibition of TLR-4-NF-κB-dependent signalling.
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Linfocitos B/efectos de los fármacos , Isquemia Encefálica/tratamiento farmacológico , Ketamina/farmacología , Midazolam/farmacología , FN-kappa B/antagonistas & inhibidores , Propofol/farmacología , Receptor Toll-Like 4/antagonistas & inhibidores , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Linfocitos B/metabolismo , Isquemia Encefálica/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Hipnóticos y Sedantes/farmacología , Hipnóticos y Sedantes/uso terapéutico , Ketamina/uso terapéutico , Masculino , Midazolam/uso terapéutico , FN-kappa B/metabolismo , Neuroprotección/efectos de los fármacos , Neuroprotección/fisiología , Propofol/uso terapéutico , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Receptor Toll-Like 4/metabolismoRESUMEN
BACKGROUND: General anesthesia in patients with or at risk for neuronal injury remains challenging due to the controversial influence of volatile anesthetics on neuronal damage. We hypothesized that isoflurane, sevoflurane, and desflurane would exert variable degrees of neurotoxicity in vitro and in vivo via activation of the p75 neurotrophin receptor (p75). METHODS: SH-SY5Y cells were exposed to oxygen-glucose deprivation (OGD, 16 hours), preceded or followed by incubation with isoflurane, sevoflurane, or desflurane (1.2 minimal alveolar concentration, 2 hours). Neuronal cell death was analyzed by flow cytometry (mitochondrial membrane potential, Annexin V/propidium iodide [AV/Pi]) and quantification of lactate dehydrogenase release. We analyzed NF-κB activity by DNA-binding ELISA and luciferase assay. The role of p75 was studied using the p75-blocking peptide TAT-pep5 and siRNA knockdown. The effect of isoflurane ±p75 inhibition on retinal ischemia-reperfusion injury (IRI) in adult Sprague-Dawley rats was assessed by analyzing retinal ganglion cell (RGC) density. RESULTS: Isoflurane but not sevoflurane or desflurane postexposure aggravated OGD-induced neuronal cell death (AV/Pi positive cells: OGD 41.1% [39.0/43.3] versus OGD + isoflurane 48.5% [46.4/63.4], P = 0.001). Isoflurane significantly increased NF-κB DNA-binding and transcriptional activity of NF-κB (relative Luminescence Units: OGD 500 [499/637] versus OGD + isoflurane 1478 [1363/1643], P = 0.001). Pharmacological inhibition or siRNA knockdown of p75 counteracted the aggravating effects of isoflurane. Isoflurane increased RGC damage in vivo (IRI 1479 RGC/mm(2) [1311/1697] versus IRI + isoflurane 1170 [1093/1211], P = 0.03), which was counteracted by p75-inhibition via TAT-pep5 (P = 0.02). CONCLUSIONS: Isoflurane but not sevoflurane or desflurane postexposure aggravates neurotoxicity in preinjured neurons via activation of p75 and NF-κB. These findings may have implications for the choice of volatile anesthetic being used in patients with or at risk for neuronal injury, specifically in patients with a stroke or history of stroke and in surgical procedures in which neuronal injury is likely to occur, such as cardiac surgery and neurovascular interventions.
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Anestésicos por Inhalación/toxicidad , Isoflurano/análogos & derivados , Éteres Metílicos/toxicidad , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Receptores de Factor de Crecimiento Nervioso/metabolismo , Daño por Reperfusión/inducido químicamente , Enfermedades de la Retina/inducido químicamente , Animales , Sitios de Unión , Muerte Celular/efectos de los fármacos , Hipoxia de la Célula , Línea Celular Tumoral , Desflurano , Relación Dosis-Respuesta a Droga , Femenino , Glucosa/deficiencia , Humanos , Isoflurano/toxicidad , Masculino , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Neuronas/metabolismo , Neuronas/patología , Fragmentos de Péptidos/farmacología , Regiones Promotoras Genéticas , Interferencia de ARN , Ratas Sprague-Dawley , Receptores de Factores de Crecimiento , Receptores de Factor de Crecimiento Nervioso/antagonistas & inhibidores , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Sevoflurano , Transducción de Señal/efectos de los fármacos , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacologíaRESUMEN
PURPOSE OF REVIEW: Conflicting reports about adverse events following nitrous oxide (N(2)O) application have spurred a discussion whether N(2)O should be abandoned from clinical practice. Concurrently, N(2)O is increasingly used as a single anesthetic agent in medical procedures. This article reviews and discusses reports about the present use of N(2)O. RECENT FINDINGS: Multiple publications demonstrate an increasing use of N(2)O as a procedural analgesic and sedative. Results from the Evaluation of Nitrous Oxide in the Gas Mixture for Anesthesia trial have been contrasted by recent studies reporting no increased risk for perioperative complications, particularly related to the cardiovascular and cerebrovascular system. Recent studies show that electroencephalogram-based anesthesia depth monitoring is not compatible with the use of N(2)O because of its distinct influence on electroencephalogram wave patterns. The clinical relevance of the proposed neurotoxicity, immunosuppression and influence on methionine metabolism remains unclear. Recently, its acute and long-term analgesic potency has been proven. Occupational exposure might pose a relevant health hazard. SUMMARY: Based on the present literature, abolishment of N(2)O is controversial. When avoided in patients at risk for adverse events, N(2)O is still a valuable supplement to general anesthesia and a potent procedural analgesic drug. In the latter, its use by nonanesthesiologists should be discouraged.
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Anestésicos por Inhalación , Óxido Nitroso , Atención Perioperativa , Analgésicos , Anestesiología , Anestésicos por Inhalación/efectos adversos , Anestésicos por Inhalación/farmacología , Ansiolíticos , Homocisteína/metabolismo , Humanos , Sistema Inmunológico/efectos de los fármacos , Despertar Intraoperatorio , Metionina/metabolismo , Óxido Nitroso/efectos adversos , Óxido Nitroso/farmacología , Exposición Profesional/efectos adversosRESUMEN
BACKGROUND: The heme oxygenase-1 (HO-1) enzyme pathway is of crucial importance in the removal of toxic blood components and regulation of neuroinflammation following hemorrhagic stroke. Although a circadian pattern dependency in the incidence and severity of hemorrhagic stroke exists, it is unknown whether the activity of the HO-1 system in the context of hemorrhagic injury also exhibits circadian dependency. We hypothesized that the circadian regulation of microglial HO-1 would determine the extent of neuroinflammation and neuronal injury in a murine model of subarachnoid hemorrhage (SAH). METHODS: In vitro expression patterns of HO-1 and circadian rhythm genes were analyzed in the microglial BV-2 cell line and primary microglia (PMG) using Western blot and qPCR. PMG isolated from Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl mice were used to evaluate the role of microglial HO-1. We further investigated the in vivo relevance in a murine subarachnoid hemorrhage (SAH) model using Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl mice with myeloid cell HO-1 deficiency, inducing SAH at different zeitgeber (ZT) times and analyzing the expression of HO-1 and the circadian control gene Period-2 (Per-2), respectively. Furthermore, we measured the inflammatory cytokine Monocyte Chemoattractant Protein-1 (MCP-1) in the cerebrospinal fluid of SAH patients in correlation with clinical outcome. RESULTS: HO-1 baseline expression and response to CO with blood exposure depended on ZT. In vitro expression of circadian control genes was de-synchronized in LyzM-Cre-Hmox1fl/fl PMG and did not respond to exogenous CO exposure. We found that circadian rhythm plays a crucial role in brain damage after SAH. At ZT2, we observed less phagocytic function, more vasospasm and increased microglial activation. CO reduced mortality at ZT12 in HO-1 deficient mice and reduced the difference between ZT2 and ZT12 in the inflammatory response. Induction of MCP-1 in the CSF from SAH patients was time-dependent and correlated with the expression of circadian control genes, SAH severity, functional impairment and delirium. CONCLUSIONS: Our data point towards a crucial role for the HO-1 enzyme system and circadian control in neuronal injury after a hemorrhagic stroke.
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Despite recent advances in cancer immunotherapy, certain tumor types, such as Glioblastomas, are highly resistant due to their tumor microenvironment disabling the anti-tumor immune response. Here we show, by applying an in-silico multidimensional model integrating spatially resolved and single-cell gene expression data of 45,615 immune cells from 12 tumor samples, that a subset of Interleukin-10-releasing HMOX1+ myeloid cells, spatially localizing to mesenchymal-like tumor regions, drive T-cell exhaustion and thus contribute to the immunosuppressive tumor microenvironment. These findings are validated using a human ex-vivo neocortical glioblastoma model inoculated with patient derived peripheral T-cells to simulate the immune compartment. This model recapitulates the dysfunctional transformation of tumor infiltrating T-cells. Inhibition of the JAK/STAT pathway rescues T-cell functionality both in our model and in-vivo, providing further evidence of IL-10 release being an important driving force of tumor immune escape. Our results thus show that integrative modelling of single cell and spatial transcriptomics data is a valuable tool to interrogate the tumor immune microenvironment and might contribute to the development of successful immunotherapies.
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Neoplasias Encefálicas/inmunología , Glioblastoma/inmunología , Interleucina-10/metabolismo , Células Mieloides/metabolismo , Linfocitos T/inmunología , Adulto , Anciano , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Comunicación Celular/inmunología , Línea Celular Tumoral , Femenino , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Voluntarios Sanos , Hemo-Oxigenasa 1/metabolismo , Humanos , Inmunoterapia/métodos , Inhibidores de las Cinasas Janus/farmacología , Inhibidores de las Cinasas Janus/uso terapéutico , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/metabolismo , Masculino , Persona de Mediana Edad , Neocórtex/citología , Neocórtex/inmunología , Neocórtex/patología , Cultivo Primario de Células , RNA-Seq , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Análisis de la Célula Individual , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Técnicas de Cultivo de Tejidos , Escape del Tumor , Microambiente Tumoral/inmunologíaRESUMEN
PURPOSE: Retinal ischemia/reperfusion (I/R) injury plays an important role in the pathophysiology of various ocular diseases. Retinal ganglion cells (RGCs) are particularly vulnerable to ischemia. Hydrogen sulfide (H(2)S) was recently shown to be neuroprotective in the brain and retina due to its antiapoptotic effects. Rapid preconditioning of retinal neurons by inhaled H(2)S before I/R injury may reduce apoptosis in the rat retina. METHODS: I/R injury was created on the left eye of rats (n=8) with or without inhaled H(2)S preconditioning (80 ppm) for one hour before ischemia. Densities of fluorogold prelabeled RGCs were analyzed 7 days after injury in retinal whole mounts. Retinal tissue was harvested to analyze protein expression of heat shock protein (HSP)-90 and the mitogen-activated protein kinases (MAPKs) c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)1/2 and p38 to elucidate a possible pathway of neuroprotection. DNA binding activity of the transcription factors nuclear factor-kappa-light-chain-enhancer of activated B-cells (NF-κB), cyclic adenosine monophosphate response element binding protein (CREB), and heat shock element (HSE), as well as caspase-3 cleavage and activity, were determined. Retinal sections were further assessed using anti-glial fibrillary acidic protein staining. RESULTS: RGC death after I/R injury decreased by 41.5% after H(2)S preconditioning compared to room air (p<0.001). H(2)S inhalation before ischemia reduced caspase-3 cleavage (p<0.001) and attenuated caspase-3 activity (p<0.001). Furthermore, HSP-90 expression was significantly elevated in the retina after H(2)S preconditioning. NF-κB but not CREB or HSE showed specific, H2S-dependent regulation, as well as the MAPKs ERK1/2 and JNK but not p38. CONCLUSIONS: H(2)S preconditioning mediates antiapoptotic effects in retinal I/R injury, thus exhibiting neuroprotection. Based on these observations, H(2)S could represent a novel and promising therapeutic agent to counteract neuronal injuries in the eye. Further studies are needed to prove H(2)S's neuroprotective propensity using a postconditioning approach.
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Apoptosis/efectos de los fármacos , Sulfuro de Hidrógeno/administración & dosificación , Precondicionamiento Isquémico/métodos , Fármacos Neuroprotectores/administración & dosificación , Daño por Reperfusión/fisiopatología , Vasos Retinianos/fisiopatología , Administración por Inhalación , Animales , Inhibidores de Caspasas , Muerte Celular/efectos de los fármacos , ADN/metabolismo , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Retina/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Vasos Retinianos/efectos de los fármacos , Factores de Tiempo , Distribución TisularRESUMEN
Intensive care unit (ICU)-acquired delirium is associated with adverse outcome in trauma patients with concomitant traumatic brain injury (TBI), but diagnosis remains challenging. Quantifying circadian disruption by analyzing expression of the circadian gene period circadian regulator 2 (PER2) and heme oxygenase 1 (HO1), which determines heme turnover, may prove to be potential diagnostic tools. Expression of PER2 and HO1 was quantified using qPCR from blood samples 1 day and 7 days after trauma. Association analysis was performed comparing mRNA expression levels with parameters of trauma (ISS-injury severity score), delirium, acute kidney injury (AKI) and length of ICU stay. 48 polytraumatized patients were included (equal distribution of TBI versus non-TBI) corrected for ISS, age and gender using a matched pairs approach. Expression levels of PER2 and HO1 were independent of age (PER2: P = 0.935; HO1: P = 0.988), while expression levels were significantly correlated with trauma severity (PER2: P = 0.009; HO1: P < 0.001) and longer ICU length of stay (PER2: P = 0.018; HO1: P < 0.001). High expression levels increased the odds of delirium occurrence (PER2: OR = 4.32 [1.14-13.87]; HO1: OR = 4.50 [1.23-14.42]). Patients with TBI showed a trend towards elevated PER2 (OR = 3.00 [0.84-9.33], P = 0.125), but not towards delirium occurrence (P = 0.556). TBI patients were less likely to develop AKI compared to non-TBI (P = 0.022). Expression levels of PER2 and HO1 correlate with the incidence of delirium in an age-independent manner and may potentially improve diagnostic algorithms when used as delirium biomarkers.Trial registration: German Clinical Trials Register (Trial-ID DRKS00008981; Universal Trial Number U1111-1172-6077; Jan. 18, 2018).
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Lesiones Traumáticas del Encéfalo/sangre , Delirio/sangre , Hemo-Oxigenasa 1/sangre , Proteínas Circadianas Period/sangre , Adulto , Anciano , Biomarcadores/sangre , Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/patología , Ritmo Circadiano/genética , Femenino , Regulación de la Expresión Génica/genética , Humanos , Tiempo de Internación , Masculino , Persona de Mediana Edad , Proteínas Circadianas Period/genética , Factores de Riesgo , Sueño/genética , Investigación Biomédica TraslacionalRESUMEN
We previously found that argon exerts its neuroprotective effect in part by inhibition of the toll-like receptors (TLR) 2 and 4. The downstream transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa B (NF-κB) are also affected by argon and may play a role in neuroprotection. It also has been demonstrated that argon treatment could mitigate brain damage, reduce excessive microglial activation, and subsequently attenuate brain inflammation. Despite intensive research, the further exact mechanism remains unclear. In this study, human neuroblastoma cells were damaged in vitro with rotenone over a period of 4 hours (to mimic cerebral ischemia and reperfusion damage), followed by a 2-hour post-conditioning with argon (75%). In a separate in vivo experiment, retinal ischemia/reperfusion injury was induced in rats by increasing intraocular pressure for 1 hour. Upon reperfusion, argon was administered by inhalation for 2 hours. Argon reduced the binding of the transcription factors signal transducer and activator of transcription 3, nuclear factor kappa B, activator protein 1, and nuclear factor erythroid 2-related factor 2, which are involved in regulation of neuronal damage. Flow cytometry analysis showed that argon downregulated the Fas ligand. Some transcription factors were regulated by toll-like receptors; therefore, their effects could be eliminated, at least in part, by the TLR2 and TLR4 inhibitor oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). Argon treatment reduced microglial activation after retinal ischemia/reperfusion injury. Subsequent quantitative polymerase chain reaction analysis revealed a reduction in the pro-inflammatory cytokines interleukin (IL-1α), IL-1ß, IL-6, tumor necrosis factor α, and inducible nitric oxide synthase. Our results suggest that argon reduced the extent of inflammation in retinal neurons after ischemia/reperfusion injury by suppression of transcription factors crucial for microglial activation. Argon has no known side effects or narcotic properties; therefore, therapeutic use of this noble gas appears ideal for treatment of patients with neuronal damage in retinal ischemia/reperfusion injury. The animal experiments were approved by the Commission for Animal Care of the University of Freiburg (approval No. 35-9185.81/G14-122) on October 19, 2012.
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Circadian rhythm gene expression in cerebral pacemaker regions is regulated by a transcriptional-translational feedback loop across the 24-h day-night cycle. In preclinical models of subarachnoid hemorrhage (SAH), cyclic gene expression is disrupted. Stabilization of circadian rhythm gene expression attenuates susceptibility to ischemic damage in both neuronal and myocardial tissues. In this clinical observational study, circadian rhythm gene Period-2 (Per2) mRNA expression levels were determined from blood leukocytes and cerebrospinal fluid (CSF) cells via real-time PCR on days 1, 7 and 14 after aneurysm rupture in 49 patients with spontaneous SAH. CSF Per2 expression was markedly suppressed immediately after SAH and remained suppressed over the course of two weeks of ICU treatment. Short-term mortality as well as occurrence of delirium was associated with greater extent of Per2 suppression on day 1 after SAH. Patients that developed delayed cerebral ischemia exhibited comparatively lower Per2 expression levels on day 7 after SAH, while presence of vasospasm remained unaffected. However, Per2 expression did not differ in patient groups with favourable or non-favourable functional neurological outcome (modified Rankin Scales 1-3 vs. 4-6). While our findings suggest a potential protective effect of stable circadian rhythm gene expression on the extent of ischemic damage, this effect was confined to the early disease course and was not reflected in patients' functional neurological outcome.
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(1) Background: A detailed understanding of the pathophysiology of hemorrhagic stroke is still missing. We hypothesized that expression of heme oxygenase-1 (HO-1) in microglia functions as a protective signaling pathway. (2) Methods: Hippocampal HT22 neuronal cells were exposed to heme-containing blood components and cell death was determined. We evaluated HO-1-induction and cytokine release by wildtype compared to tissue-specific HO-1-deficient (LyzM-Cre.Hmox1 fl/fl) primary microglia (PMG). In a study involving 46 patients with subarachnoid hemorrhage (SAH), relative HO-1 mRNA level in the cerebrospinal fluid were correlated with hematoma size and functional outcome. (3) Results: Neuronal cell death was induced by exposure to whole blood and hemoglobin. HO-1 was induced in microglia following blood exposure. Neuronal cells were protected from cell death by microglia cell medium conditioned with blood. This was associated with a HO-1-dependent increase in monocyte chemotactic protein-1 (MCP-1) production. HO-1 mRNA level in the cerebrospinal fluid of SAH-patients correlated positively with hematoma size. High HO-1 mRNA level in relation to hematoma size were associated with improved functional outcome at hospital discharge. (4) Conclusions: Microglial HO-1 induction with endogenous CO production functions as a crucial signaling pathway in blood-induced inflammation, determining microglial MCP-1 production and the extent of neuronal cell death. These results give further insight into the pathophysiology of neuronal damage after SAH and the function of HO-1 in humans.