C1 neurons mediate a stress-induced anti-inflammatory reflex in mice

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Abstract

C1 neurons, located in the medulla oblongata, mediate adaptive autonomic responses to physical stressors (for example, hypotension, hemorrhage and presence of lipopolysaccharides). We describe here a powerful anti-inflammatory effect of restraint stress, mediated by C1 neurons: protection against renal ischemia-reperfusion injury. Restraint stress or optogenetic C1 neuron (C1) stimulation (10 min) protected mice from ischemia-reperfusion injury (IRI). The protection was reproduced by injecting splenic T cells that had been preincubated with noradrenaline or splenocytes harvested from stressed mice. Stress-induced IRI protection was absent in Chrna7 knockout (a7nAChR−/−) mice and greatly reduced by destroying or transiently inhibiting C1. The protection conferred by C1 stimulation was eliminated by splenectomy, ganglionic-blocker administration or β2-adrenergic receptor blockade. Although C1 stimulation elevated plasma corticosterone and increased both vagal and sympathetic nerve activity, C1-mediated IRI protection persisted after subdiaphragmatic vagotomy or corticosterone receptor blockade. Overall, acute stress attenuated IRI by activating a cholinergic, predominantly sympathetic, anti-inflammatory pathway. C1s were necessary and sufficient to mediate this effect.

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Figure 1: Restraint stress protects against renal IRI.
Figure 2: Adoptive transfer of splenocytes protects against renal IRI.
Figure 3: C1 stimulation protects against renal IRI.
Figure 4: C1 neurons mediate the protective effect of restraint stress against renal IRI.
Figure 5: Corticosterone is released by C1 stimulation and restraint stress but plays no detectable role in protecting kidneys from renal IRI.
Figure 6: C1 stimulation activates both divisions of the autonomic nervous system.
Figure 7: Protection against renal IRI by C1 stimulation is mediated via the autonomic nervous system and the spleen.
Figure 8: Restraint stress and C1 stimulation produce opposite effects on AP and HR.

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Acknowledgements

Editorial comments by D.A. Bayliss (University of Virginia, Pharmacology Department) are gratefully acknowledged. We thank the University of Virginia Research Histology Core for their assistance in preparation of histology slides. Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the NIH under award numbers RO1HL028785 and RO1HL074011 (to P.G.G.), by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (NIH) under award numbers R01DK085259 and R01DK062324 (to M.D.O.) and by two Japan Society for the Promotion of Science Postdoctoral Fellowships for Overseas Researchers (awarded separately to C.A. and T.I.). The stereology data described here was performed with an MBF Bioscience and Zeiss microscope system for stereology and tissue morphology funded by National Institutes of Health grant 1S10RR026799-01 (to M.D.O.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

C.A., T.I., D.L.R., R.L.S., M.D.O. and P.G.G. designed research studies; C.A., T.I., M.A.I., K.E.V., L.H. and H.Y. conducted experiments and acquired and analyzed the data; and C.A., T.I., D.L.R., R.L.S., M.D.O. and P.G.G. wrote the manuscript.

Correspondence to Patrice G Guyenet.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Protective effect of restraint stress: histology

Representative kidney histological sections (H&E stain) illustrating the protective effect of restraint stress against renal ischemia-reperfusion injury (IRI) in DBH-cre mice. IRI induced cast formation, tubule dilation, and/or tubular epithelial denucleation, and these alterations were less severe in DBH-cre mice with restraint stress. Scale bar: 100 μm for main panels and 50 μm for insets.

Supplementary Figure 2 C1 stimulation activates breathing in conscious DBH-cre mice

(a) Rostrocaudal distribution of mCherry-immunoreactive catecholaminergic (TH+) neurons in the ventrolateral medulla oblongata of 25 DBH-cre mice 5-6 weeks after unilateral injection of AAV2–DIO–EF1α–ChR2–mCherry (neurons per section vs. distance from bregma in mm). (b) Location of fiber optic tips. Distance caudal to bregma in mm shown at lower left of each section. Scale bar 500 μm (c) Original plethysmography recordings (top trace, respiratory flow signal, inspiration downward; bottom trace, respiratory frequency (fR). Optogenetic stimulation of the C1 neurons (5, 10, 15, and 20 Hz; pulse duration, 10 ms; 10 s train at blue bars) activated breathing in a DBH-cre mouse (d-f) Effect of C1 neuron stimulation on fR (d), tidal volume (Vt, e), and minute volume (Ve, f) in 27 DBH-cre mice. Statistics: one-way repeated measure ANOVA with Tukey–Kramer test; [F(3, 104) = 128.1, P < 0.0001] (d), [F(3, 104) = 12.01, P < 0.0001] (e), and [F(3, 104) = 87.49, P < 0.0001] (f). *** P < 0.001 vs. 5 Hz, ††† P < 0.001 vs. 10 Hz, and ‡‡‡ P < 0.001 vs. 20 Hz.

Supplementary Figure 3 Prolonged C1 stimulation regularizes the breathing rate and produces quiescence in conscious DBH-cre mice

(a) Plethysmography record of a DBH-cre mouse prior to and during 10 min unilateral photostimulation of C1 cells (frequency, 5 Hz; pulse duration, 10 ms). Top trace, air flow signal; bottom trace, respiratory frequency (fR). Large amplitude signals in the flow trace denote locomotor activity or other motor behavior (grooming, sniffing). Incidence of such events was much reduced during C1 cell stimulation in DBH-cre mice. Higher resolution respiratory flow trace during active behavior (a1), spontaneous quiescence (a2), and quiescence induced by C1 neuron stimulation (a3). (b) Representative Poincaré plots of breathing frequency (instantaneous frequency of (n+1)th breath vs. previous breath (nth)) before (left) and during C1 neuron stimulation (right panel; 5 Hz; pulse duration, 10 ms, 10 min) in DBH-cre mouse. Standard deviations normal to the line of identity (SD1, sqrt(Σ(fRn+1-fRn)^2 /2(N-1))) and along the line of identity (SD2, sqrt(Σ(fRn+1+fRn-2m)^2 /2(N-1))) were calculated using LabView (National Instruments) and the ellipse area was calculated (SD1 x SD2 x π). (c) Mean ellipse area was significantly reduced by C1 neuron stimulation indicating more frequent episodes of quiescence (10 min; frequency, 5 Hz; pulse duration, 10 ms) (n = 6). Statistical analysis: unpaired t test [t(10) = 3.41, P = 0.0067] ** P < 0.01 vs. Laser(-).

Supplementary Figure 4 Protective effect of C1 stimulation: histology

Representative histological sections (H&E stain) of kidney illustrating the protective effect of C1 neuron stimulation in DBH-cre mice against kidney ischemia-reperfusion injury (IRI). IRI induced cast formation, tubule dilation, and/or tubular epithelial denucleation. These alterations were reduced by C1 photostimulation (ChR2-mcherry expressing vector) but not by sham stimulation (mCherry-expressing vector). Scale bar: 100 μm for main panels and 50 μm for insets. Laser: 5 Hz, 10 min. IR, ischemia-reperfusion.

Supplementary Figure 5 Histological evidence of successful subdiaphragmatic vagotomy

Representative histological picture of the Fluoro-Gold (FG) expression in dorsal motor nucleus of the vagus (DMV). Sham (n = 3) and subdiaphragmatic vagotomized (sVNX, n = 3) mice received i.p. injections of FG (5 mL/kg of 1% solution in sterile water). After 3 days, the mice were deeply anesthetized and perfused. FG expression at DMV is observed in Sham mouse but not in sVNX mouse. Scale bar: 500 μm for main panels and 250 μm for insets.

Supplementary Figure 6 Renal hypoxia-inducible factor 1α mRNA level after 10 min of ischemia vs. 10 min of C1 stimulation

Kidney Hif1α/Gapdh mRNA ratio was significantly elevated after renal ischemia-reperfusion in DBH-cre mice (10 min of complete renal vessel clamping) whereas optogenetic activation of C1 cells (5 Hz for 10 min) produced no change in mRNA level. Kidneys were harvested immediately after 10 min of renal ischemia or 10 min after the cessation of C1 photostimulation. Statistics (5 mice per group): one-way ANOVA with Tukey–Kramer test; [F(2, 20) = 5.653, P = 0.0186]. * P < 0.05 vs. IR(-):Laser(-) and IR(+):Laser(-).

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Abe, C., Inoue, T., Inglis, M. et al. C1 neurons mediate a stress-induced anti-inflammatory reflex in mice. Nat Neurosci 20, 700–707 (2017) doi:10.1038/nn.4526

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