Non-invasive vagus nerve stimulation in a hungry state decreases heart rate variability.

Vagus nerve signals from the gut to brain carry information about nutrients and drive food reward. Such signals are disrupted by consuming large amounts of high-calorie foods, necessitating greater food intake to elicit a similar neural response. Non-invasive vagus nerve stimulation (nVNS) via a branch innervating the ear is a candidate treatment for obesity in humans. There is disagreement on the optimal location of nVNS in the ear for experimental and clinical studies. There are also no studies comparing nVNS in hungry and post-prandial states. We aimed to compare ear position(s) for nVNS and explore the effects of nVNS during hungry and post-prandial states on proxies for autonomic outflow (heart-rate variability) and efferent metabolism (gastric wave frequency and resting energy expenditure). In a within-subject design, 14 participants (10 women, on average 29.4 +/- 6.7 years old) received nVNS in four different locations (cymba conchae, tragus, earlobe, or tragus AND cymba conchae) on separate days. In each session, participants were asked to consume a palatable chocolate flavored milk. With electrography on the abdomen and indirect calorimetry in a canopy, we measured electro-cardiogram, electro-gastrogram and resting energy expenditure for 15 min before and at least 35 min after consumption of the palatable drink. We also collected ratings of the palatable drink and internal and other states. Pre-drink consumption (in a hungry state) we observed no differences in the effect of location of acute nVNS on resting energy expenditure and gastric wave measures. However, nVNS in cymba conchae decreases heart-rate variability (relative to sham) and ratings of how much participants want to consume the drink (relative to tragus AND cymba conchae and a trend relative to sham). After drink consumption and with continued nVNS, gastric wave frequency is unchanged, and resting energy expenditure increases regardless of stimulation location. Heart-rate variability decreases in all locations, except cymba conchae. We also observe a trend for an increase in gastric wave amplitude in late post-drink consumption time-points in cymba conchae. We observe no support for the combined stimulation of tragus AND cymba conchae being more effective than either of the individual locations. These results suggest that nVNS in the cymba conchae in a hungry state has a similar acute effect on vagal tone as food consumption: to decrease heart rate variability. This effect then negates the usual postprandial effects of a decrease in heart rate variability as seen in the other nVNS locations. These preliminary observations suggest that nVNS in cymba conchae may act primarily on vagal afferent autonomic (and only modestly on metabolic output) in a similar way as food consumption does.

Integrating macrophages into organotypic co-cultures: a 3D in vitro model to study tumor-associated macrophages.

Tumor progression is controlled by signals from cellular and extra-cellular microenvironment including stromal cells and the extracellular matrix. Consequently, three-dimensional in vitro tumor models are essential to study the interaction of tumor cells with their microenvironment appropriately in a biologically relevant manner. We have previously used organotypic co-cultures to analyze the malignant growth of human squamous cell carcinoma (SCC) cell lines on a stromal equivalent in vitro. In this model, SCC cell lines are grown on a collagen-I gel containing fibroblasts. Since macrophages play a critical role in the progression of many tumor types, we now have expanded this model by integrating macrophages into the collagen gel of these organotypic tumor co-cultures. This model was established as a murine and a human system of skin SCCs. The effect of macrophages on tumor progression depends on their polarization. We demonstrate that macrophage polarization in organotypic co-cultures can be modulated towards and M1 or an M2 phenotype by adding recombinant IFN-γ and LPS or IL-4 respectively to the growth medium. IL-4 stimulation of macrophage-containing cultures resulted in enhanced tumor cell invasion evidenced by degradation of the basement membrane, enhanced collagenolytic activity and increased MMP-2 and MMP-9. Interestingly, extended co-culture with tumor cells for three weeks resulted in spontaneous M2 polarization of macrophages without IL-4 treatment. Thus, we demonstrate that macrophages can be successfully integrated into organotypic co-cultures of murine or human skin SCCs and that this model can be exploited to analyze macrophage activation towards a tumor supporting phenotype.