RESUMO
OBJECTIVE: While it has been shown that different T-cell subsets have a detrimental role in the acute phase of ischemic stroke, data on the impact of dendritic cells (DC) are missing. Classic DC can be characterized by the cluster of differentiation (CD)11c surface antigen. METHODS: In this study, we depleted CD11c+ cells by using a CD11c-diphtheria toxin (DTX) receptor mouse strain that allows selective depletion of CD11c+ cells by DTX injection. For stroke induction, we used the model of transient middle cerebral artery occlusion (tMCAO) and analyzed stroke volume and functional outcome on days 1 and 3 as well as expression of prototypical pro- and anti-inflammatory cytokines on day 1 after tMCAO. Three different protocols for CD11c+ cell depletion, tMCAO duration, and readout time point were applied. RESULTS: Injection of DTX (5 or 100 ng/g) reliably depleted CD11c+ cells without influencing the fractions of other immune cell subsets. CD11c+ cell depletion had no impact on stroke volume, but mice with a longer DTX pretreatment performed worse than those with vehicle treatment. CD11c+ cell depletion led to a decrease in cortical interleukin (IL)-1ß and IL-6 messenger ribonucleic acid levels. CONCLUSIONS: We show, for the first time, that CD11c+ cell depletion does not influence stroke volume in a mouse model of focal cerebral ischemia. Nevertheless, given the unspecificity of the CD11c surface antigen for DC, mouse models that allow a more selective depletion of DC are needed to investigate the role of DC in stroke pathophysiology.
Assuntos
Antígeno CD11c/deficiência , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/genética , Infarto da Artéria Cerebral Média/patologia , Animais , Antígeno CD11c/genética , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Toxina Diftérica/administração & dosagem , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Despite the efficacy of neuroprotective approaches in animal models of stroke, their translation has so far failed from bench to bedside. One reason is presumed to be a low quality of preclinical study design, leading to bias and a low a priori power. In this study, we propose that the key read-out of experimental stroke studies, the volume of the ischemic damage as commonly measured by free-handed planimetry of TTC-stained brain sections, is subject to an unrecognized low inter-rater and test-retest reliability with strong implications for statistical power and bias. As an alternative approach, we suggest a simple, open-source, software-assisted method, taking advantage of automatic-thresholding techniques. The validity and the improvement of reliability by an automated method to tMCAO infarct volumetry are demonstrated. In addition, we show the probable consequences of increased reliability for precision, p-values, effect inflation, and power calculation, exemplified by a systematic analysis of experimental stroke studies published in the year 2015. Our study reveals an underappreciated quality problem in translational stroke research and suggests that software-assisted infarct volumetry might help to improve reproducibility and therefore the robustness of bench to bedside translation.