Decreased neuroinflammation correlates to higher vagus nerve activity fluctuations in near-term ovine fetuses: a case for the afferent cholinergic anti-inflammatory pathway?

Frasch et al. Journal of Neuroinflammation (2016) 13:103 DOI 10.1186/s12974-016-0567-x RESEARCH Open Access Decreased neuroinflammation correlates to higher vagus nerve activity fluctuations in near-term ovine fetuses: a case for the afferent cholinergic anti-inflammatory pathway? M. G. Frasch1,2,3,4,8*, M. Szynkaruk4, A. P. Prout4, K. Nygard5, M. Cao1,2, R. Veldhuizen6, R. Hammond7 and B. S. Richardson4,6 Abstract Background: Neuroinflammation in utero may contribute to brain injury resulting in life-long neurological disabilities. The pivotal role of the efferent cholinergic anti-inflammatory pathway (CAP) in controlling inflammation, e.g., by inhibiting the HMGB1 release, via the macrophages’ α7 nicotinic acetylcholine receptor (α7nAChR) has been described in adults, but its importance in the fetus is unknown. Moreover, it is unknown whether CAP may also exert anti-inflammatory effects on the brain via the anatomically predominant afferent component of the vagus nerve. Methods: We measured microglial activation in the ovine fetal brain near term 24 h after the umbilical cord occlusions mimicking human labor versus controls (no occlusions) by quantifying HMGB1 nucleus-to-cytosol translocation in the Iba1+ and α7nAChR+ microglia. Based on multiple clinical studies in adults and our own work in fetal autonomic nervous system, we gauged the degree of CAP activity in vivo using heart rate variability measure RMSSD that reflects fluctuations in vagus nerve activity. Results: RMSSD correlated to corresponding plasma IL-1β levels at R = 0.57 (p = 0.02, n = 17) and to white matter microglia cell counts at R = −0.89 (p = 0.03). The insult increased the HMGB1 translocation in α7nAChR+ microglia in a brain region-dependent manner (p < 0.001). In parallel, RMSSD at 1 h post insult correlated with cytosolic HMGB1 of thalamic microglia (R = −0.94, p = 0.005), and RMSSD at pH nadir correlated with microglial α7nAChR in the white matter (R = 0.83, p = 0.04). Overall, higher RMSSD values correlated with lower HMGB1 translocation and higher α7nAChR intensity per area in a brain region-specific manner. Conclusions: Afferent fetal CAP may translate increased vagal cholinergic signaling into suppression of cerebral inflammation in response to near-term hypoxic acidemia as might occur during labor. Our findings suggest a new control mechanism of fetal neuroinflammation via the vagus nerve, providing novel possibilities for its non-invasive monitoring in utero and for targeted treatment. Keywords: Fetus, Labor, Vagus, Microglia, HMGB1, CHRNA7, HRV, RMSSD * Correspondence: 1 Department of Obstetrics and Gynaecology, CHU Ste-Justine Research Centre, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada 2 Department of Neurosciences, CHU Ste-Justine Research Centre, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada Full list of author information is available at the end of the article © 2016 Frasch et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Frasch et al. Journal of Neuroinflammation (2016) 13:103 Background Induced animal sepsis and clinical-pathologic studies in adults indicate that loss of the cholinergic antiinflammatory pathway’s (CAP) inhibitory influence unleashes innate immunity, producing higher levels of proinflammatory mediators that exacerbate tissue damage. This decrease in CAP activity also decreases short-term heart rate variability (HRV), e.g., as measured by the beat-to-beat HRV measures, such as root mean square of successive differences in R-R intervals of ECG (RMSSD), a measure of vagal modulation of HRV [1, 2]. Thus, short-term HRV measures reflect CAP activity in adults [3]. Of note, RMSSD also reflects vagal activity in fetal sheep [4]. Increased CAP vagal activity inhibits the release of pro-inflammatory cytokines such as interleukin (IL)-1β [1]. This systemic CAP effect is mediated via the α7 nicotinic acetylcholine receptor (α7nAChR) expressed on macrophages [5]. However, recent studies have shown a similar α7nAChR-dependent effect in brain microglia in vitro [6–8]. In adult species, high-mobility group box protein 1 (HMGB1), a non-histone DNA-binding protein, acts as an important pro-inflammatory cytokine linking necrosis with ensuing inflammation by translocating from the neuronal nucleus to the cytosol and then to the extracellular space, leading to microglial activation [9]. Much attention has been paid to the effects of α7nAChR stimulation on HMGB1 secretion because of its therapeutic potential to treat sepsis; HMGB1 represents a crucial link between neuronal necrosis and the cerebral inflammatory response mediated by microglia, thus impacting the long-term outcome of neurological injury [9, 10]. HMGB1 also acts as a potent pro-inflammatory cytokine when secreted by microglia in response to inflammatory stimuli [11]. This requires translocation of HMGB1 from nucleus to cytosol [9]. Systemic and neuroinflammation have been implicated as important pathophysiological mechanisms acting independently to cause fetal brain injury or contributing to hypoxic-asphyxial brain injury with consequences for postnatal health [12, 13]. In the late-gestation ovine and human fetus, the autonomic nervous system and cholinergic vagal activity in particular are known to be sufficiently mature [2, 14]. We have shown that CAP is active spontaneously near term, such that individual baseline RMSSD values and the levels of the pro-inflammatory cytokines IL-1β and IL-6 are inversely correlated, reflecting spontaneous CAP activity [15]. First, we hypothesized that the fetal inflammatory response induced by hypoxic acidemia will result in an increase of systemic CAP activity as a compensatory mechanism and an inhibitory effect of CAP on the Page 2 of 14 cerebral inflammatory response. The systemic inflammatory response will be reflected by an increased vagal activity and hence a correlation of RMSSD and IL-1β. Second, we sought to determine the effect of fetal hypoxic-acidemia insult on brain regional activation of the microglia expressing α7nAChR, and the relation of systemic and cerebral CAP activation to the intracellular HMGB1 localization in these cells. Thus, we hypothesized that the cerebral inflammatory response will result in microglial HMGB1 translocation from the nucleus to the cytosol due to increased microglial activation via α7nAChR and this HMGB1 translocation will correlate with the degree of CAP (...truncated)