RESUMEN
Bioluminescence imaging (BLI) relies on the biochemical reaction between substrate and enzyme that triggers light emission upon convergence. Here, we present a protocol to study molecular oxygen dynamics in the in vivo mouse brain using the oxygen-dependent reaction between luciferase and its substrate. We describe steps for acute craniotomy, viral transfection, substrate administration, imaging, and analysis of hypoxic pockets. This protocol offers superior spatiotemporal properties compared to established approaches like electrodes and phosphorescence. For complete details on the use and execution of this protocol, please refer to Beinlich et al.1.
RESUMEN
Consciousness is lost within seconds upon cessation of cerebral blood flow. The brain cannot store oxygen, and interruption of oxidative phosphorylation is fatal within minutes. Yet only rudimentary knowledge exists regarding cortical partial oxygen tension (Po2) dynamics under physiological conditions. Here we introduce Green enhanced Nano-lantern (GeNL), a genetically encoded bioluminescent oxygen indicator for Po2 imaging. In awake behaving mice, we uncover the existence of spontaneous, spatially defined "hypoxic pockets" and demonstrate their linkage to the abrogation of local capillary flow. Exercise reduced the burden of hypoxic pockets by 52% compared with rest. The study provides insight into cortical oxygen dynamics in awake behaving animals and concurrently establishes a tool to delineate the importance of oxygen tension in physiological processes and neurological diseases.
Asunto(s)
Corteza Cerebral , Circulación Cerebrovascular , Hipoxia Encefálica , Mediciones Luminiscentes , Saturación de Oxígeno , Oxígeno , Animales , Ratones , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/metabolismo , Oxígeno/sangre , Oxígeno/metabolismo , Presión Parcial , Hipoxia Encefálica/sangre , Hipoxia Encefálica/diagnóstico por imagen , Hipoxia Encefálica/metabolismo , Vasodilatación , Mediciones Luminiscentes/métodos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipercapnia/sangre , Hipercapnia/diagnóstico por imagen , Hipercapnia/metabolismoRESUMEN
Entire Helicobacter Pylori Neutrophil Activated Protein (HPNAP) and its truncated forms NH(2)-terminal region HPNAP(1-57) and C-terminal region HPNAP(58-144) after cloning into pET29c vector, purification and removal of LPS traces were subjected to human neutrophil activation. Our results revealed that the C-terminal region of HPNAP is indispensable for human neutrophil stimulation and their further adhesion to endothelial cells - a step necessary to H. pylori inflammation - in a ratio equal to that exhibited by the entire protein. In addition, experiments concerning the implication of Arabino-Galactan-Proteins (AGPs) derived from Chios Mastic Gum (CMG), the natural resin of the plant Pistacia lentiscus var. Chia revealed the inhibition of neutrophil activation and therefore their adhesion to endothelial cells, in vitro. Both, the involvement of HPNAP C-terminal region in stimulation-adhesion of neutrophils to endothelial cells as well as the inhibition of this process by AGPs have to be further investigated and may be exploited in a future anti-inflammatory therapy for H. pylori patients.
