Full Assessment of Lung Mechanics Using Computer-Controlled, Forced Oscillation Technique.

The forced oscillation technique (FOT) is a powerful and accurate method to quantify the mechanical properties of the airways and tissues of the respiratory system. Here we provide a detailed protocol for the measurement of mouse respiratory mechanical parameters. We present a procedure for mouse endotracheal intubation using a handcrafted intubation platform and confirmation module. The FlexiVentFX™ system (Scireq Inc.) is utilized for the thorough assessment of lung function with the FlexiWare™ software serving as a unit for the planning, experimentation, and analysis. The protocol has been standardized and adapted for use by our center for lung-function phenotyping of mouse models generated for the International Mouse Phenotyping Consortium (IMPC). The simplified steps, technical considerations, and integrated hardware-software demonstration make this protocol adaptable and implementable for researchers interested in using FOT for lung-function evaluation. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Support Protocol 1: Assembly of the FlexiVentFX™ system for measurements Support Protocol 2: FlexiWare database management Support Protocol 3: A guide for the construction of intubation platform and confirmation module Basic Protocol 1: Mouse endotracheal intubation Basic Protocol 2: Assessment of mouse basal lung function.

Ablation of Nampt in AgRP neurons leads to neurodegeneration and impairs fasting- and ghrelin-mediated food intake.

Agouti-related protein (AgRP) neurons in the arcuate nucleus of the hypothalamus regulates food intake and whole-body metabolism. NAD+ regulates multiple cellular processes controlling energy metabolism. Yet, its role in hypothalamic AgRP neurons to control food intake is poorly understood. Here, we aimed to assess whether genetic deletion of nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in NAD+ production, affects AgRP neuronal function to impact whole-body metabolism and food intake. Metabolic parameters during fed and fasted states, and upon systemic ghrelin and leptin administration were studied in AgRP-specific Nampt knockout (ARNKO) mice. We monitored neuropeptide expression levels and density of AgRP neurons in ARNKO mice from embryonic to adult age. NPY cells were used to determine effects of NAMPT inhibition on neuronal viability, energy status, and oxidative stress in vitro. In these cells, NAD+ depletion reduced ATP levels, increased oxidative stress, and promoted cell death. Agrp expression in the hypothalamus of ARNKO mice gradually decreased after weaning due to progressive AgRP neuron degeneration. Adult ARNKO mice had normal glucose and insulin tolerance, but exhibited an elevated respiratory exchange ratio (RER) when fasted. Remarkably, fasting-induced food intake was unaffected in ARNKO mice when evaluated in metabolic cages, but fasting- and ghrelin-induced feeding and body weight gain decreased in ARNKO mice when evaluated outside metabolic cages. Collectively, deletion of Nampt in AgRP neurons causes progressive neurodegeneration and impairs fasting and ghrelin responses in a context-dependent manner. Our data highlight an essential role of Nampt in AgRP neuron function and viability.